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

Summary

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.

Abstract

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's progression and facilitate the development of potential anticancer drugs.

Introduction

Among all oncological disorders, lung cancer not only inflicts the highest life loss but also claims the second-highest number of new patients every year in the US¹. This devastating malignancy stands as a major obstacle in modern healthcare, urging for a deeper understanding of its intricate biology and more efficacious therapeutic modalities². Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer and tends to develop into solid tumors³. One of the foremost challenges in lung cancer is the dynamic and heterogeneous tumor microenvironment, which profoundly influences the cancer's progression and responses to therapeutic interventions^4,5,6. A deeper understanding of the interplay between cancer cells and their microenvironment at different stages of NSCLC calls for refined pathological models that recapitulate the histological features of NSCLC progression.

In this regard, orthotopic animal models emerge as a promising avenue for NSCLC research. Unlike commonly employed subcutaneous xenograft models⁷, orthotopic models feature cancer cells that are directly inoculated into the organ of origin. For lung cancer, this means implanting cancer cells directly into the lung tissue^8,9. Consequently, orthotopic models of lung cancer better mimic the native tumor microenvironment, including the neighboring tissues, vessels, and immune components, thus improving their physiological and clinical relevance.

Three-dimensional multicellular spheroids (MCS) represent another promising approach to recapitulating features of the tumor environment. Most cancers are characterized by their complex tumor microenvironment, including the various cell-cell interactions, the extracellular matrix, and the gradients in oxygen and nutrients^10,11. Traditional 2D cell cultures lack the spatial and structural complexity to recapitulate these tumor-specific features¹². In contrast, MCS of appropriate size feature a heterogeneous structure with a hypoxic and necrotic core, which recapitulates not only the intratumoral microenvironment but also the physiological barrier against drug penetration, which is a major mechanism of drug resistance in anticancer therapy^13,14,15.

Taking advantage of both the orthotopic animal models and the MCS culturing techniques, MCS have been inoculated to immune-compromised mice to successfully construct orthotopic models of breast cancer and prostate cancer^16,17. Herein, we report the detailed methodology to construct and characterize a murine orthotopic xenograft model of lung cancer. This method employs the intrapulmonary inoculation of 3D MCS derived from fluorescent human lung cancer cells (A549-iRFP)¹⁸. This model offers an exceptional opportunity to observe the in vivo progression of lung cancer through stages that closely parallel the four clinical stages of NSCLC. Furthermore, the xenograft cancer of this model responded to the clinically established antilung cancer drug, cisplatin.

Protocol

The animal study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the University of the Pacific (Animal Protocols 19R10 and 22R10). Eight Male athymic nude mice aged 5-6 weeks, weighing 20-25 g, bred with the referenced Rodent Diet and housed under pathogen-free (SPF) conditions, were used for the present study. Cages, bedding, and drinking water were autoclaved and changed regularly. A schematic of tumor inoculation in mice is shown in Figure 1. See the Table of Materials for details related to all materials and instruments used in this protocol.

1. Establishment of three-dimensional MCS of A549-iRFP cells

1. Incubate A549-iRFP cells (human lung adenocarcinoma) in a humidified atmosphere with 5% CO₂ at 37 °C and grow them in complete growth medium DMEM containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1 µg/mL puromycin.
2. Seed A549-iRFP cells into 96-well ultra-low attachment, round-bottom spheroid microplate at a seeding density of 4,000 cells/well, using 100 µL of complete growth medium supplemented with 0.3% collagen.
3. Centrifuge the microplates at 300 × g for 7 min at 4 °C to facilitate the MCS formation. Examine the MCS morphology under a microscope to ensure cell aggregation after centrifugation.
4. After 48 h, add 100 µL of complete growth medium to each well to achieve a total volume of 200 µL of growth medium per well.
5. Replace 100 µL of growth medium in each well with fresh complete growth medium every other day.

2. Characterization of A549-iRFP MCS

1. Observe and image the MCS morphology using a microscope and estimate the MCS volume by simulation with the referenced software from MATLAB based on the phase-contrast microscope images.
   1. Open one MCS image in ImageJ software.
   2. Use Freehand selections to select the edge of the MCS and add it to ROI Manager.
   3. Click Edit | Selection | Create Mask.
   4. Click Edit | Invert.
   5. Click File | Save As | Tiff.
   6. Open ReViSP software.
   7. Open the saved TIFF image by clicking Browse.
   8. Click Start to complete the MCS simulation.
2. Measure the iRFP fluorescent signal using the infrared imaging system at the 700 nm channel and quantify the fluorescent signal using software (e.g., Image Studio).
   1. Open Image Studio software.
   2. In the Channels tab, select 700 and Intensity = 5.
   3. In the Scan Controls tab, select 84 µm, Medium, 0.0 mm, and Flip Image. Keep everything else as default.
   4. Click Start to start scanning.
3. Assess and quantify cell viability using the 3D cell viability assay according to the vendor protocol.
4. Assess and quantify cellular protein using the BCA Assay according to the vendor protocol.

3. MCS selection for tumor inoculation

NOTE: After MCS are seeded into spheroid microplates and grown for 2-3 weeks with regular growth medium exchange, select MCS with the following appropriate characteristics for tumor inoculation.

1. Observe the MCS morphology under a microscope and choose MCS with a round shape with overall smooth edges but with 5-10 rough buddings (arrows in Figure 2F) and a diameter within the range of 700-800 µm.
2. Measure the iRFP fluorescence (refer to step 2.2) and select the MCS whose fluorescent signals are within one standard deviation of the average.

4. Intrapulmonary MCS inoculation

NOTE: Use 70% isopropyl alcohol spray to clean the surgical station and the tools before handling the animals. The surgeon should wear sterile gloves throughout the surgical procedures.

1. Anesthetize each mouse with an IP injection of 80 mg/kg ketamine and 12 mg/kg xylazine; pinch the mouse feet with forceps to ensure full anesthesia.
2. Inject 0.05 mg/kg of buprenorphine hydrochloride subcutaneously to reduce the pain.
3. Apply ophthalmic ointment on the eyes to prevent dryness.
4. Place the mouse on an infrared heat pad in a dorsal posture and secure the limbs in a stretching position with tapes.
   NOTE: Provide heat support to the anesthetized mouse throughout the whole surgical procedure.
5. Disinfect the back skin by swabbing with alternating rounds of iodine and alcohol, at least three times each.
6. Cut a 0.5-1 cm incision using surgical scissors on the left back side (Figure 1B). Carefully use forceps to separate the muscle and fat tissues until the chest wall and lung motion are visible.
7. Transfer one selected MCS from a microplate into a glass Petri dish containing ice-cold PBS, using a pipette.
8. Attach a 20 G needle onto a 100 µL glass syringe, precool them on ice, and draw 20 µL of a precooled mixture of PBS and Matrigel (1:1 v/v).
9. Use the syringe to quickly aspirate one MCS from the Petri dish in a minimum volume of PBS-Matrigel mixture, keeping the MCS in the metal part of the needle.
   NOTE: Keep a tight grip on the syringe and plunger to prevent the MCS from slipping out of the needle.
10. Gently insert the needle vertically between two rib bones to ~3 mm depth and slowly inject all the 20 µL PBS and Matrigel mixture that contains the MCS.
    NOTE: Avoid excessive force during the insertion to prevent damage to the lung, vasculature, or heart.
11. Carefully remove the needle and apply triple antibiotic ointment to the wound.
12. Seal the incision with surgical clips, which are to be removed after 2 weeks.
    NOTE: Do not suture the incision because the suture lines may interfere with fluorescence imaging. The nude mice are able to heal naturally after surgery.
13. Place the animal on an infrared heat pad and cover it with laboratory wipes to maintain the body temperature. Monitor the animal for at least 30-60 min until it wakes up and moves properly.
14. Inject 0.05 mg/kg of buprenorphine hydrochloride subcutaneously every 8 h for the first 48 h to reduce the pain.

5. Postsurgical monitoring

1. Measure the body weight every 3-4 days.
2. Measure the iRFP fluorescent signal from the tumor xenograft every 3-4 days on a small animal imaging system at 700 nm channel in four postures: left, right, dorsal, and ventral, while the mice are kept under anesthesia by isoflurane inhalation (flow rate: 1.5-2 L/min oxygen containing 1.5% isoflurane) over 2-3 min.
   NOTE: Use 70% isopropyl alcohol spray to clean the small animal imaging system before putting animal onto the imaging station. The surgeon should wear sterile gloves throughout the surgical procedures.
3. Quantify the net fluorescent intensity of the xenograft cancer in vivo using software (e.g., Image Studio).
   1. Use a fixed rectangle (A1, 200 x 125 pixels) as the sampling window for the chest area of the mice.
   2. Use the free-hand function to trace the shape of the mice within the rectangle (A1) and measure the fluorescent intensity (F1) in area A1.
   3. Use another smaller rectangle (A2, 25 x 40 pixels) to measure the background fluorescence (F2) of the mice in the thigh area.
   4. Use equation (1) to calculate the net fluorescent intensity of the xenograft cancer in each mouse:
           (1)

Results

Characterization of A549-iRFP MCS
A549-iRFP MCS were successfully cultured in spheroid microplates with the assistance of collagen and centrifugation. When MCS reached a diameter of approximately 500 µm after 1 week, both A549 and A549-iRFP MCS were exposed to a variety of anticancer drugs and formulations for 3 days and then maintained in drug-free growth medium for 4 additional days. The A549-iRFP MCS exhibited a response pattern closely mirroring that of the parent A549 cells. A549 and A549...

Discussion

The construction of A549-iRFP MCS is a straightforward and highly reproducible lab procedure and can be translated to MCS formation for multiple cell lines. The MCS generated with the aid of centrifugation and collagen exhibits a more integral and solid-tumor-like structure within 3-4 days. This method ensures the formation of robust spheroids that maintain their integral structure for extended periods, typically 2-3 weeks or even longer until small buddings begin to emerge. By employing centrifugation and collagen, we s...

Materials

Name	Company	Catalog Number	Comments
100 µL Glass Syringe	Hamilton	Part/REF #80601
20 G Needle	Thermo Fisher Scientific Inc.	14 826D
96-well Ultra-Low-Attachment Spheroid Microplate	Corning	15-100-173
A549-iRFP	Imanis Life Sciences	CL082-STAN
AIN-93M Mature Rodent Diet	Research Diets, Inc.	D10012M
Athymic Nude Mouse	Charles River Laboratories, Inc.	Strain Code: 490; homozygous
BCA	Pierce	23227
Buprenorphine Hydrochloride	Patterson Veterinary	NDC Number: 42023-179-05
CellTiter-Glo 3D Cell Viability Assay	Promega	G9683
Collagen	Gibco	A1064401
DMEM	Corning	MT10013CV
Fetal Bovine Serum (FBS)	Cytiva HyClone	SH3039603
ImageJ	Open source tool (https://imagej.net/ij/)	N/A
Image Studio	LI-COR	Version 5.2
Isoflurane	Patterson Veterinary	NDC Number: 17033-0091-25
Ketamine	Patterson Veterinary	NDC Number: 50989-0161-06
Microscope	Keyence	Model number: BZ-X710
Matrigel	Corning	CB-40234
Odyssey Infrared Imaging 205 System	LI-COR	Model number: 9140
PBS	Corning	MT21040CV
Pearl Trilogy small animal imaging system	LI-COR	Model number: 9430
Penicillin-Streptomycin	Corning	MT30002CI
Puromycin	Thermo Fisher Scientific Inc.	AAJ67236XF
ReViSP software from MATLAB	Open source tool on Sourceforge (https://sourceforge.net/projects/revisp/)	N/A
Surgical Clips--AutoClip System	Fine Science Tools	12020-00
Xylazine	Patterson Veterinary	NDC Number: 61133-6017-01