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&#38;D group, specifically Robert Abraham, Puja Sapra, Karen Widbin and Jennifer Tejeda for their support of the program.

1. Passaging and Culturing TUM622 Cells and CAFs in 2D Cultures
<ol>
	<li>Passaging and culturing TUM622 cells
	<ol>
		<li>Warm 3D culture medium and cell dissociation reagents (see Table of Materials) for TUM622 cells at 37 &#176;C.</li>
		<li>Passage TUM622 cells at 80% confluency in 2D flasks. Usually, this occurs 1 week after passaging.</li>
		<li>Discard old medium from a T75 flask and wash once with 6 mL of HEPES buffer. Avoid pipetting directly onto the cells.</li>
		<li>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.</li>
		<li>Add 2 mL of trypsin/EDTA and incubate at 37 &#176;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.</li>
		<li>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).</li>
		<li>Pipette up-and-down gently to further dissociate the cells using a 10 mL pipette. Transfer the suspension through a 40 &#181;m cell strainer into a 50 mL conical tube.</li>
		<li>Count cell numbers using a hemocytometer or automated cell counter.</li>
		<li>Seed 0.8 x 106 cells/T75 flask in 20 mL of 3D culture medium (see Table of Materials).</li>
		<li>Feed the cells every other day by replacing half of the spent medium with fresh medium.</li>
	</ol>
	</li>
	<li>Passaging and culturing CAFs
	<ol>
		<li>Passage CAFs when cells reach confluency. Usually, this occurs after 5 days of culturing from a 1:2 split.</li>
		<li>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 &#176;C.</li>
		<li>In a T75 flask, rinse CAFs with phosphate-buffered saline (PBS) once then add 2 mL of trypsin/EDTA and incubate at 37 &#176;C for 5 min.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Transfer the cell suspension into a 50 mL conical tube and spin down at 300 x g for 5 min at room temperature.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Plating TUM622 Cells in the Extracellular Matrix for 3D Culturing
<ol>
	<li>The day before the experiment, thaw vials of basement membrane matrix in a 4 &#176;C refrigerator overnight. Cooldown plastic pipettes (2 mL) and tips at -20 &#176;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.</li>
	<li>On the day of the experiment, warm 3D culture medium, HEPES buffer, trypsin/EDTA and trypsin neutralization buffer (TNS) in a 37 &#176;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.</li>
	<li>Cooldown the tissue culture plates on a metal platform cooler placed on ice. Place centrifuge tubes on a metal cooling rack on ice.</li>
	<li>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.</li>
	<li>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 &#176;C for 5 min.</li>
	<li>Aspirate the supernatant carefully with an aspirating pipette attached to an unfiltered tip (20 &#181;L), leaving approximately 100 &#181;L of the medium in the tube (use markings on the tube as a guide).</li>
	<li>Gently tap on the side of the tube to dislodge and dissociate the pellet before returning it to the cooling rack.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using pre-cooled tips, pipette the matrix in each tube up and down about 10 times to make a uniform cell suspension.</li>
	<li>Transfer 310 &#181;L of cell/matrix suspension into each well of a pre-cooled 24-well plate. The pipette is placed at a 90&#176; 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.</li>
	<li>To facilitate downstream immunofluorescence analysis, plate the cell/matrix suspension in parallel into 2-well chamber slides. Transfer 100 &#181;L 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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
3. 3D Coculturing of TUM622 Cells and CAFs in the Extracellular Matrix
<ol>
	<li>Prepare cell suspensions of TUM622 and CAFs according to section 2.</li>
	<li>Count the CAF cell density by taking 10 &#181;L of cell suspension and mixing it with 10 &#181;L of trypan blue.</li>
	<li>Add 10 &#181;L 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.</li>
	<li>Co-embedding TUM622 cells and CAFs in basement membrane matrix
	<ol>
		<li>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.</li>
		<li>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 &#181;L of TUM622/CAFs mix to chamber slides as described in step 2.12).</li>
	</ol>
	</li>
	<li>Coculturing TUM622 with overlaid CAFs in basement membrane matrix (see Table of Materials)
	<ol>
		<li>Set up TUM622 mono-culture according to steps 2.5-2.13.</li>
		<li>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.</li>
		<li>Aspirate the supernatant and resuspend the CAFs in 1 mL of 3D culture medium.</li>
		<li>Transfer the 1 mL of CAFs suspension to the well containing the embedded TUM622 cells.</li>
	</ol>
	</li>
</ol>
4. Harvesting TUM622 Acini for RNA/Protein Extraction and Fluorescence-activated Cell Sorting (FACS)
<ol>
	<li>Prepare wash buffer and cell harvesting buffer according to the 3D cell harvesting kit protocol the previous day and chill overnight at 4 &#176;C.</li>
	<li>Keep plates on a plate cooler and other reagents on ice before starting the extraction process.</li>
	<li>Aspirate media from 3D culture wells without touching the matrix and gently wash the well 3 times with 1 mL of wash buffer.</li>
	<li>Aspirate the final wash and add 1 mL of cell harvesting buffer to each well.</li>
	<li>Use a p1000 pipette tip to scrape the matrix off of each well.</li>
	<li>Pipette up and down to further dissociate the matrix.</li>
	<li>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.</li>
	<li>Repeat steps 4.5-4.7, and transfer all mix of the same well into one 15 mL conical tube.</li>
	<li>Cap the tubes and rock at 4 &#176;C for 30 min.</li>
	<li>Fill each tube with ice-cold PBS up to 10 mL and then centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>While spinning, prepare lysis buffer for protein and RNA collection.</li>
	<li>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.</li>
</ol>
5. Immunofluorescence of TUM622 Acini
<ol>
	<li>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 &#181;g/mL goat anti-mouse F(ab&#39;)2)</li>
	<li>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.</li>
	<li>Add pre-chilled 4% PFA to fix the acini and incubate on ice for 20 min.</li>
	<li>Remove 4% PFA and wash three times with 2 mL of pre-chilled PBS each for 5 min with gentle rocking on a rocker.</li>
	<li>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.</li>
	<li>Gently aspirate the permeabilization buffer from the chamber slide to avoid sample loss. This is achieved by adding a fine tip (20 &#181;L) to the aspirating pipette and pressing the tip towards the corner of the chamber.</li>
	<li>Wash three times with 2 mL of pre-chilled PBS each for 5 min with gentle rocking on a rocker.</li>
	<li>Block the sample with the primary blocking buffer on ice for 1 h.</li>
	<li>Remove primary blocking buffer and add secondary blocking buffer and block for 30 min.</li>
	<li>Add primary antibodies in primary blocking buffer and incubate overnight at 4 &#176;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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add PBS with DAPI (1:1,000 dilution) during the last wash to stain the nucleus. The perform another 2 washes in PBS.</li>
	<li>Image the samples on a confocal microscope within 3 days. 
	NOTE: Due to the size of the organoids and limits in the objective&#39;s working distance, samples are usually imaged at 10x or 20x magnification.</li>
</ol>
6. Preparing 3D Culture Samples for Immunohistochemistry
<ol>
	<li>Aspirate medium from 2-well chamber slides and rinse once with PBS.</li>
	<li>Fix 3D cultures in 4% PFA at 37 &#176;C overnight.</li>
	<li>Remove 4% PFA, surround cultures with 2.5 mL of histology sample gel (see Table of Materials) and place the slide at 4 &#176;C to solidify for at least 1 h.</li>
	<li>Transfer samples surrounded with histological sample gel to tissue cassettes and processed in an automated tissue sample processor overnight.</li>
	<li>Embed samples in paraffin wax and prepare for sectioning12.</li>
</ol>
7. 3D Cytotoxicity Assay for Compound Screening (Example for One 96-well Plate)
<ol>
	<li>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.</li>
	<li>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 &#181;L of basement membrane matrix. Pipette up and down a few times to allow even mixing of cells within the matrix.</li>
	<li>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.</li>
	<li>Transfer the matrix cell mixture to the cooling reservoir without creating bubbles.</li>
	<li>Using a mechanical multichannel pipette (10-300 &#181;L), transfer 70-75 &#181;L of the mix cells into each appropriate well of a 96-well plate.</li>
	<li>Incubate plate at 37 &#176;C and 5% CO2 for 30 min for the basement membrane matrix to solidify.</li>
	<li>Add 100 &#181;L of media in all rows and return the plate to the incubator.</li>
	<li>Start compound dosing the next day or later depending on the goal of the experiment.</li>
	<li>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.</li>
	<li>The number of TUM622 spheroids could be quantified using 3D imager according to the manufacturer&#39;s protocol.</li>
</ol>

<ol>
	<li>Cancer Genome Atlas Research Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genomic+characterization+of+squamous+cell+lung+cancers.">Comprehensive genomic characterization of squamous cell lung cancers.</a> Nature. 489 (7417), 519-525 (2012).</li><li>Nelson, C. M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Extracellular+Matrix,+Scaffolds,+and+Signaling:+Tissue+Architecture+Regulates+Development,+Homeostasis,+and+Cancer.">Of Extracellular Matrix, Scaffolds, and Signaling: Tissue Architecture Regulates Development, Homeostasis, and Cancer.</a> Annual Review of Cell and Developmental Biology. 22 (1), 287-309 (2006).</li><li>Quail, D. F., Joyce, J. 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The authors are employees and shareholders of Pfizer Inc.

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...

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 NSCLC6,7,8. 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 CAFs9,10. 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.

<table><tbody><tr><td>Bronchial Epithelial Growth Medium</td><td>Lonza</td><td>CC-3170</td><td>BEGM</td></tr><tr><td>Cell Strainer 40um</td><td>ThermoFisher</td><td>352340</td><td>For passing TUM622 cells</td></tr><tr><td>Cleaved Caspase 3 antibody</td><td>Cell Signaling Technology</td><td>9661 (RRID:AB_2341188)</td><td>Rabbit</td></tr><tr><td>CoolRack CFT30</td><td>Biocision</td><td>BCS-138</td><td>For 3D culture</td></tr><tr><td>CoolSink XT96F</td><td>Biocision</td><td>BCS-536</td><td>For 3D culture</td></tr><tr><td>Cultrex 3D Cell Harvesting Kit</td><td>Bio-Techne</td><td>3448-020-K</td><td></td></tr><tr><td>Cultrex (preferred for co-culture)</td><td>Bio-Techne</td><td>3443-005-01</td><td>For 3D culture</td></tr><tr><td>CXCR4 antibody</td><td>Abcam</td><td>Ab124824 (RRID:AB_10975635)</td><td>Rabbit</td></tr><tr><td>E-cadherin antibody</td><td>BD Biosciences</td><td>610182 (RRID:AB_397581)</td><td>Mouse</td></tr><tr><td>GelCount</td><td>Oxford Optronix</td><td></td><td>For Acini counts and measurements</td></tr><tr><td>GM130 antibody</td><td>BD Biosciences</td><td>610822 (RRID:AB_398141)</td><td>Mouse</td></tr><tr><td>Goat Serum</td><td>Vector Labs</td><td>S1000 (RRID:AB_2336615)</td><td>For Immunofluorescence</td></tr><tr><td>Heat-inactivated FBS</td><td>Gibco</td><td>10082-147</td><td>For CAFs</td></tr><tr><td>Histology sample gel</td><td>Richard Allan Scientific</td><td>HG-4000-012</td><td>For Immunofluorescence</td></tr><tr><td>Integrin alpha 6 antibody</td><td>Millipore Sigma</td><td>Mab1378 (RRID:AB_2128317)</td><td>Rat</td></tr><tr><td>Involucrin antibody</td><td>Abcam</td><td>Ab68 (RRID:AB_305656)</td><td>Mouse</td></tr><tr><td>Ki67 antibody</td><td>Abcam</td><td>Ab15580 (RRID:AB_443209)</td><td>Rabbit</td></tr><tr><td>Lab-Tec II chambered #1.5 German Coverglass System</td><td>Nalge Nunc International</td><td>155379 (2)</td><td>For 3D culture</td></tr><tr><td>Lab-Tec II chambered #1.5 German Coverglass System</td><td>Nalge Nunc International</td><td>155409 (8)</td><td>For 3D culture</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td>For CAFs</td></tr><tr><td>Matrigel (preferred for mono-culture)</td><td>Corning</td><td>356231</td><td>For 3D culture</td></tr><tr><td>p63 antibody</td><td>Cell Signaling Technology</td><td>13109 (SRRID:AB_2637091)</td><td>Rabbit</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td>For CAFs</td></tr><tr><td>ReagentPack Subculture Reagents</td><td>Lonza</td><td>CC-5034</td><td>For TUM622 cell dissociation</td></tr><tr><td>RPMI</td><td>ThermoFisher</td><td>11875-093</td><td>For CAFs</td></tr><tr><td>Sox2 antibody</td><td>Cell Signaling Technology</td><td>3579 (RRID:AB_2195767)</td><td>Rabbit</td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12604-021</td><td>For CAF dissociation</td></tr><tr><td>Vi-Cell</td><td>Bechman Coulter</td><td></td><td>Automatic cell counter</td></tr><tr><td>Vimentin antibody</td><td>Abcam</td><td>Ab92547 (RRID:AB_10562134)</td><td>Rabbit</td></tr><tr><td>&amp;beta;-catenin antibody</td><td>Cell Signaling Technology</td><td>2677s (RRID:AB_1030943)</td><td>Mouse</td></tr></tbody></table>

multidimensional coculture system to model lung squamous carcinoma progression

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.

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...

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Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

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 ...

multidimensional-coculture-system-to-model-lung-squamous-carcinoma

Research

JoVE Journal

Cancer Research

7.1K Views.  Pfizer Worldwide Research and Development. 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.

Video: Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicine

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioengineering

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

Author Spotlight: Establishing a Murine Non-Small Cell Lung Cancer Model for Developing Nanoformulations of Anticancer Drugs

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Renal Cancer and Healthy Tissue Co-Culture Using Microphysiological Systems

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

3D Co-culture of Lung Cancer Cells with CAFs: An In Vitro Model System to Study Tumor Progression

Source: Chen, S., et al. <a href="https://www.jove.com/v/60644/multidimensional-coculture-system-to-model-lung-squamous-carcinoma">Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression.</a>&#160;J. Vis. Exp.&#160;(2020)
This video describes the two invitro model systems to capture the tissue architectural changes during lung squamous carcinoma progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts. This system provides a unique platform to investigate the role of intrinsic and extrinsic changes that affect the tumor phenotype.

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

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A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Three-dimensional Co-culture Model for Tumor-stromal Interaction

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