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 tissue architecture². During this process, proper apical-basal cell polarity, cell-cell and cell-matrix contacts are lost, permitting uncontrolled growth and invasive behavior of the tumor cells. It is now widely appreciated that the malignant features of cancer cells cannot be manifested without an important interplay between cancer cells and their local tumor microenvironment (TME)³. Key components in the TME including extracellular matrix (ECM), cancer-associated fibroblasts (CAFs) as well as endothelial cells and infiltrating immune cells actively shape the TME and drives tumorigenesis⁴. Nevertheless, our current understanding of how the tumor cells and these key components in the TME interact to drive tissue architectural changes during LUSC progression is very limited.

Three-dimensional (3D) culture is an important tool to study the biological activities of cell-intrinsic and extrinsic changes in regulating tissue architectural changes in both normal and diseased tissues⁵. 3D cultures provide the appropriate structural and functional context that is usually lacking in traditional two-dimensional (2D) cultures. The added dimensions of such systems more closely mimic tissue in vivo in many aspects of cell physiology and cellular behaviors, including proliferation, differentiation, migration, protein expression and response to drug treatment. In recent years, efforts from various labs have led to the development of in vitro 3D models for both the normal lung as well as NSCLC^6,7,8. However, a model for lung squamous carcinoma that can recapitulate both the dynamic tissue architectural changes during tumorigenesis as well as incorporate key stromal components was unavailable.

Here, we describe the methods for establishing a novel 3-dimensional (3D) coculture system using primary PDX-derived LUSC cells (termed TUM622) and CAFs^9,10. Both TUM622 and CAFs are derived from NSCLC patient with poorly differentiated tumors¹⁰. When embedded as single cells in ECM, a rare subpopulation of TUM622 cells have the capacity to form organoids with acinar-like structures that display proper apical-basal cell polarity. These acinar-like structures are hyperplastic, display heterogeneous expression of stem-like and differentiation markers similar to the original tumor while remaining non-invasive, and thus mimic the earliest stage of LUSC development. Importantly, we showed that the tissue architecture of the acinar-like structures could be altered by inhibition of cell-intrinsic signaling pathways with small molecule inhibitors or addition of key components in the ECM such as CAFs, the latter of which enhances acini formation and further provokes the acini to become invasive when in close proximity. Together, these data suggest that this 3D co-culture system of LUSC organoids provides a valuable platform for the investigation of the dynamic reciprocity between LUSC cells and the TME and could be adapted for monitoring the response of LUSC cells to drug treatment¹¹.

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 buffer. Add 4 mL of trypsin/EDTA (0.25 mg/mL, see Table of Materials) for a quick rinse and discard the trypsin/EDTA.
   5. Add 2 mL of trypsin/EDTA and incubate at 37 °C for 5 min. Remove flasks from the incubator and tap the flasks to loosen the cells without creating air bubbles and return flasks to the incubator for an additional 5 min.
      NOTE: Prolonged exposure to trypsin will irreversibly damage the cells and alter their phenotype, thus it is recommended to limit the time cells are exposed to trypsin.
   6. Confirm cells have detached and dissociated under a light microscope (4x or 10x). Add 4 mL of neutralization buffer (TNS buffer) (see subculture reagent information in the Table of Materials) followed by 10 mL of 3D culture medium (see Table of Materials).
   7. Pipette up-and-down gently to further dissociate the cells using a 10 mL pipette. Transfer the suspension through a 40 µm cell strainer into a 50 mL conical tube.
   8. Count cell numbers using a hemocytometer or automated cell counter.
   9. Seed 0.8 x 10⁶ cells/T75 flask in 20 mL of 3D culture medium (see Table of Materials).
   10. Feed the cells every other day by replacing half of the spent medium with fresh medium.
2. Passaging and culturing CAFs
   1. Passage CAFs when cells reach confluency. Usually, this occurs after 5 days of culturing from a 1:2 split.
   2. Prepare CAF medium using RPMI basal medium with 20% heat-inactivated fetal bovine serum, 1% L-Glutamine and 1% Penicillin/Streptomycin. Warm the medium to 37 °C.
   3. In a T75 flask, rinse CAFs with phosphate-buffered saline (PBS) once then add 2 mL of trypsin/EDTA and incubate at 37 °C for 5 min.
   4. Observe under a light microscope to ensure cells have dissociated in the flask (4x or 10x). If not, extend the incubation for another 2-3 min.
   5. Once cells have detached and dissociated, add 10 mL of 3D culture medium to neutralize the trypsin/EDTA and pipette up and down several times to further dissociate the CAFs.
   6. Transfer the cell suspension into a 50 mL conical tube and spin down at 300 x g for 5 min at room temperature.
   7. Discard the supernatant and resuspend the pellet in an appropriate volume of 3D culture medium (see Table of Materials) and passage into two new T75 flasks.

2. Plating TUM622 Cells in the Extracellular Matrix for 3D Culturing

1. The day before the experiment, thaw vials of basement membrane matrix in a 4 °C refrigerator overnight. Cooldown plastic pipettes (2 mL) and tips at -20 °C overnight.
   NOTE: Not all lots of basement membrane matrix have the same capacity to support the 3D growth of TUM622 cells. Therefore, it is necessary to acquire and test multiple lots of basement membrane matrix to identify those that support robust acini formation. Usually, this requires a higher protein concentration (16-18 mg/mL) in the matrix.
2. On the day of the experiment, warm 3D culture medium, HEPES buffer, trypsin/EDTA and trypsin neutralization buffer (TNS) in a 37 °C water bath. Immediately before setting up the culture, take the thawed basement membrane matrix out of the fridge and put the vial on ice.
3. Cooldown the tissue culture plates on a metal platform cooler placed on ice. Place centrifuge tubes on a metal cooling rack on ice.
4. Using TUM622 cells obtained from step 1.1.7, calculate the desired number of cells needed for plating. Typically, 15,000-30,0000 cells are needed per well of a 24-well plate. Lower density is more suited for imaging and quantification, while higher density is preferred when collecting cells for RNA extraction or western blotting.
5. Transfer cell suspension into a cooled centrifuge tube (each tube containing cells for triplicate plating) and spin down at 300 x g in a hanging bucket centrifuge at 4 °C for 5 min.
6. Aspirate the supernatant carefully with an aspirating pipette attached to an unfiltered tip (20 µL), leaving approximately 100 µL of the medium in the tube (use markings on the tube as a guide).
7. Gently tap on the side of the tube to dislodge and dissociate the pellet before returning it to the cooling rack.
8. Using the 2 mL pre-cooled pipettes, gently mix the matrix by pipetting up and down a few times while keeping the vial in contact with the ice. Pipette at an even and moderate speed so that no bubbles are introduced into the matrix during this procedure.
9. Transfer the appropriate volume of the matrix into each centrifuge tube. For plating triplicates in a 24-well plate, add 1.1 mL of basement membrane matrix to each tube.
10. Using pre-cooled tips, pipette the matrix in each tube up and down about 10 times to make a uniform cell suspension.
11. Transfer 310 µL of cell/matrix suspension into each well of a pre-cooled 24-well plate. The pipette is placed at a 90° angle to the plate surface and the suspension added to the center of the well. The suspension should spread and cover the entire well without needing to tilt the plate.
12. To facilitate downstream immunofluorescence analysis, plate the cell/matrix suspension in parallel into 2-well chamber slides. Transfer 100 µL of cell/matrix suspension into the center of a well of 2-well chamber slide (see Table of Materials). This allows the matrix to form a dome-like structure with much smaller volume.
13. Return the plate and the chamber slide back into a tissue culture incubator and incubate for 30 min to allow the matrix to solidify. Examine the plate/slide under a light microscope to ensure that single cells are evenly distributed within the matrix (4x or 10x).
14. Add 1 mL of pre-warmed 3D culture complete medium into each well and 1.5 mL of 3D culture medium to each well of the chamber slide then return them to the incubator.

3. 3D Coculturing of TUM622 Cells and CAFs in the Extracellular Matrix

1. Prepare cell suspensions of TUM622 and CAFs according to section 2.
2. Count the CAF cell density by taking 10 µL of cell suspension and mixing it with 10 µL of trypan blue.
3. Add 10 µL of the mixture to each of the two chambers on a hemacytometer to count and calculate cell density.
   NOTE: CAFs have irregular shapes and may not be accurately counted on an automatic cell counter.
4. Co-embedding TUM622 cells and CAFs in basement membrane matrix
   1. Based on the cell density information, calculate the desired number of cells used for plating. CAFs are seeded at a 2:1 ratio of TUM622 cells. For example, for 30,000 TUM622 cells seeded, 60,000 CAFs are co-embedded.
   2. Transfer the appropriate volume of TUM622 as well as CAFs cell suspension into the same centrifuge tube and follow steps 2.5-2.11 for plating into 24-well plates. For immunofluorescence, transfer 60 µL of TUM622/CAFs mix to chamber slides as described in step 2.12).
5. Coculturing TUM622 with overlaid CAFs in basement membrane matrix (see Table of Materials)
   1. Set up TUM622 mono-culture according to steps 2.5-2.13.
   2. Transfer twice the number of CAFs suspension (compared to the number of TUM622 cells seeded) into a centrifuge tube and spin down at 300 x g for 5 min at room temperature.
   3. Aspirate the supernatant and resuspend the CAFs in 1 mL of 3D culture medium.
   4. Transfer the 1 mL of CAFs suspension to the well containing the embedded TUM622 cells.

4. Harvesting TUM622 Acini for RNA/Protein Extraction and Fluorescence-activated Cell Sorting (FACS)

1. Prepare wash buffer and cell harvesting buffer according to the 3D cell harvesting kit protocol the previous day and chill overnight at 4 °C.
2. Keep plates on a plate cooler and other reagents on ice before starting the extraction process.
3. Aspirate media from 3D culture wells without touching the matrix and gently wash the well 3 times with 1 mL of wash buffer.
4. Aspirate the final wash and add 1 mL of cell harvesting buffer to each well.
5. Use a p1000 pipette tip to scrape the matrix off of each well.
6. Pipette up and down to further dissociate the matrix.
7. Transfer 1 mL of the mix to a pre-chilled 15 mL conical tube. Add another 1 mL of harvesting buffer to the same well.
8. Repeat steps 4.5-4.7, and transfer all mix of the same well into one 15 mL conical tube.
9. Cap the tubes and rock at 4 °C for 30 min.
10. Fill each tube with ice-cold PBS up to 10 mL and then centrifuge at 300 x g for 5 min at 4 °C.
11. Aspirate the supernatant without touching the pellet. The supernatant should contain matrix fragments, but the spheroids should all be collected at the bottom of the tube.
12. Add ice-cold PBS for a second wash. Invert the tube a few times to dissociate the pellet. Spin down at 300 x g for 5 min.
13. While spinning, prepare lysis buffer for protein and RNA collection.
14. Carefully aspirate the supernatant and add lysis buffer for downstream processing to collect protein or RNA. Alternatively, cells could be resuspended for flow analysis/FACS sorting or serial passaging.

5. Immunofluorescence of TUM622 Acini

1. Prepare immunofluorescence buffer (IF buffer: PBS with 0.1% bovine serum albumin (BSA), 0.2% Triton X-100 and 0.05% Tween-20), primary blocking buffer (IF buffer with 10% goat serum), secondary blocking buffer (primary blocking buffer with 20 µg/mL goat anti-mouse F(ab')₂)
2. Aspirate medium from 2-well chamber slides, rinse once with PBS and set the slide on metal plate cooler on ice. The chamber slide should remain on the metal plate cooler for the remainder of the protocol.
3. Add pre-chilled 4% PFA to fix the acini and incubate on ice for 20 min.
4. Remove 4% PFA and wash three times with 2 mL of pre-chilled PBS each for 5 min with gentle rocking on a rocker.
5. Aspirate PBS and permeabilize with 1.5 mL of 0.5% Triton X-100 in PBS (pre-chilled) for 20 min. By the end of this procedure, the dome-like structure will become loose.
6. Gently aspirate the permeabilization buffer from the chamber slide to avoid sample loss. This is achieved by adding a fine tip (20 µL) to the aspirating pipette and pressing the tip towards the corner of the chamber.
7. Wash three times with 2 mL of pre-chilled PBS each for 5 min with gentle rocking on a rocker.
8. Block the sample with the primary blocking buffer on ice for 1 h.
9. Remove primary blocking buffer and add secondary blocking buffer and block for 30 min.
10. Add primary antibodies in primary blocking buffer and incubate overnight at 4 °C.
    NOTE: The concentration of the antibodies used here should be higher than normally used for staining cells in 2D culture. Most of the primary antibodies used in this study are diluted at 1:100 dilution (see Table of Materials).
11. Remove primary antibodies and wash the sample 3 times with 2 mL of cold IF buffer.
    NOTE: The samples could be loose, take extra caution when aspirating.
12. Incubate the samples in secondary antibodies diluted in primary blocking buffer for 1 h at RT. The preferred secondary antibodies should be highly cross-adsorbed to reduce background staining. Most secondary antibodies used in this study are diluted at 1:200 dilution.
13. Remove secondary antibodies and wash the sample 3 times with 2 mL of cold IF buffer.
    NOTE: The samples could be loose, take extra caution when aspirating.
14. Add PBS with DAPI (1:1,000 dilution) during the last wash to stain the nucleus. The perform another 2 washes in PBS.
15. Image the samples on a confocal microscope within 3 days.
    NOTE: Due to the size of the organoids and limits in the objective's working distance, samples are usually imaged at 10x or 20x magnification.

6. Preparing 3D Culture Samples for Immunohistochemistry

1. Aspirate medium from 2-well chamber slides and rinse once with PBS.
2. Fix 3D cultures in 4% PFA at 37 °C overnight.
3. Remove 4% PFA, surround cultures with 2.5 mL of histology sample gel (see Table of Materials) and place the slide at 4 °C to solidify for at least 1 h.
4. Transfer samples surrounded with histological sample gel to tissue cassettes and processed in an automated tissue sample processor overnight.
5. Embed samples in paraffin wax and prepare for sectioning¹².

7. 3D Cytotoxicity Assay for Compound Screening (Example for One 96-well Plate)

1. Set a 96-well plate on a plate cooler, a 25 mL reservoir on a reservoir cooler and a 15 mL conical tubes on ice before starting the experiment.
2. Prepare cell matrix suspensions of TUM622 cells in a pre-chilled 15 mL polypropylene conical tube by adding the appropriate volume of basement membrane matrix to cells. The desired density for TUM622 cells is 10,000 cells per 70-75 µL of basement membrane matrix. Pipette up and down a few times to allow even mixing of cells within the matrix.
3. Move the plate with plate cooler and reservoir with a reservoir cooler away from the ice to a dry surface to avoid contact of basement membrane matrix with ice during transfer.
4. Transfer the matrix cell mixture to the cooling reservoir without creating bubbles.
5. Using a mechanical multichannel pipette (10-300 µL), transfer 70-75 µL of the mix cells into each appropriate well of a 96-well plate.
6. Incubate plate at 37 °C and 5% CO₂ for 30 min for the basement membrane matrix to solidify.
7. Add 100 µL of media in all rows and return the plate to the incubator.
8. Start compound dosing the next day or later depending on the goal of the experiment.
9. Spheroids can be re-fed and re-dosed every 2-3 days for up to 10 days, by removing spent media with an 8- or 12-well vacuum manifold and replacing with fresh media with or without desired compounds.
10. The number of TUM622 spheroids could be quantified using 3D imager according to the manufacturer's protocol.

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...

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