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

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.

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 10 µL of buprenorphine hydrochloride (0.3 mg/mL) subcutaneously to reduce the pain.
3. Apply ophthalmic ointment on the eyes to prevent dryness.
4. Place the mouse in a dorsal posture and secure the limbs in a stretching position with tapes.
5. Sterilize the back skin with iodine and alcohol swab sticks.
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 10 µL of buprenorphine hydrochloride (0.3 mg/mL) subcutaneously 3x a day 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.
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)

Representative 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-iRFP MCS showed similar dose-response curves and IC₅₀ values to cisplatin, one of the first-line anticancer drugs against lung cancer (Figure 2A). The viability of A549 and A549-iRFP MCS was inhibited to similar degrees by other anticancer drugs or formulations (Figure 2B).

The A549-iRFP MCS were monitored over a period of up to 6 weeks by iRFP fluorescence, cell viability, and cellular protein levels. The volume of MCS was estimated by software simulation based on the phase-contrast microscope images, and the simulated morphology exhibited similar shapes to those imaged by microscope and fluorescent signal (Figure 2C). The cell viability of A549-iRFP MCS was quantified by 3D cell viability assay, and the assay was validated with MCS of various sizes from 350 to 950 µm in diameter. There were no significant differences observed across various volume ratios between the growth medium containing MCS and the assay reagent, and between different shaking times of 5 minutes and 10 minutes. (Supplementary Figure S1). All four biomarkers increased with time, with larger variations at later time points (Figure 2D). The iRFP fluorescence exhibited strong correlations with the other three more traditional quantitative measurements of MCS: volume (Pearson r = 0.9307, p < 0.0001), cell viability (Pearson r = 0.7666, p < 0.0001), and cellular protein (Pearson r = 0.7317, p < 0.0001) (Figure 2E).

Tumor progression in mice
After growing for 2-3 weeks, A549-iRFP MCS with appropriate characteristics, including morphology, MCS diameter, appropriately rough edges, and fluorescent signal (for detailed parameters, see protocol section 3), were selected for tumor inoculation. After the tumor inoculation surgery, the body weight of tumor-bearing mice was measured every 3 days and presented as the change ratio compared to the weight on Day 0 right before surgery. The mouse body weight decreased slightly after surgery and recovered quickly within 1 week. Without treatment, the body weight increased gradually until ~Day 40 but decreased drastically (~15%) in the following week, leading to the euthanasia (three-fold anesthetic overdose: 80 mg/kg ketamine and 12 mg/kg xylazine via IP injection) of mice according to IACUC guidelines (Figure 3A). Tumor-bearing mice were imaged every 3 days in four postures: left, dorsal, right, and ventral sides. Among these, the left (where the tumor is inoculated) and ventral (where both sides of the lungs can be observed) were selected as key postures for the quantification of net fluorescent intensity according to equation (1) (Figure 3B,C). The net fluorescence intensity from both postures showed a similar trend of tumor progression, with fluorescence from the ventral side growing slightly slower than the left side.

The MCS xenograft in this orthotopic model progressed through distinct phases mirroring the four clinical stages of NSCLC (Figure 3D)^18,19,20. Around Day 8 post-MCS inoculation, concentrated fluorescence emerged in the left lung, signifying the establishment of a localized tumor in line with Stage 1 NSCLC in clinical settings. By approximately Day 11, the fluorescence notably intensified and/or dispersed across multiple sites in the left lung, indicating the presence of tumor(s) resembling Stage 2 NSCLC. Subsequently, at approximately Day 18, increased fluorescent signals were observed from the ventral side, suggestive of Stage 3-like progression of the xenograft. Moreover, anatomical examination and ex vivo imaging revealed tumor growth on the surface of the heart, a significant hallmark of Stage 3 NSCLC. By approximately Day 27 post-MCS inoculation, perfused fluorescence appeared on both sides of the lung, and open-chest anatomy disclosed perfused tumor growth in the heart, trachea, and major blood vessels, indicative of cancer metastasis akin to Stage 4 NSCLC.

Response of tumor-bearing mice to cisplatin
A pilot efficacy study was conducted on this orthotopic lung cancer murine model. Ten days after tumor inoculation, 5 mg/kg cisplatin or normal saline was injected via the tail vein of tumor-bearing mice, one injection per week, totaling two injections. The ventral-side images on tumor-bearing mice are presented in Figure 4A. The body weight and tumor fluorescence were monitored every 3-4 days. Compared with the normal saline group, the mice in the cisplatin group lost significant body weight after each cisplatin injection but recovered in the weeks after the cisplatin treatment (Figure 4B). The net fluorescence intensity in the cisplatin group showed a slower trend of increase than that in the saline group (Figure 4C). At the end of the in vivo fluorescence monitoring, the animals were sacrificed and their lungs dissected and imaged by fluorescence. After normalization by the total tissue protein, the fluorescent intensity of the lungs dissected from mice in the cisplatin group was confirmed to be lower than that of the normal saline group. (Figure 4E). In the ventral fluorescent imaging (Figure 4A), the images in the red rectangles indicate the first time when the tumor fluorescence was visualized in both the left and the right lungs, which is a key indicator of tumor metastasis at Stage IV NSCLC. Cisplatin, one of the first-line lung cancer drugs, slowed down the tumor metastasis from the left lung to the right lung (Figure 4D).

Figure 1: Flow chart of the study. (A) Seeding A549-iRFP cells into spheroid microplates to form MCS with the assistance of collagen and centrifugation. (B) Intrapulmonary inoculation of A549-iRFP MCS with appropriate characteristics into a mouse's left lung to construct the orthotopic lung cancer xenograft model, mimicking the four stages of lung cancer in clinical settings. The white syringe containing one MCS indicates tumor inoculation while the two red syringes indicate drug treatment administered through the tail vein. Abbreviation: MCS = multicellular spheroid. Please click here to view a larger version of this figure.

Figure 2: Characterization of A549-iRFP MCS. (A) Dose-response curves of A549-iRPF MCS and A549 MCS to cisplatin after 3-day exposure and 4-day drug-free growth (Mean ± SD, N = 3-4). (B) Cell viability of A549-iRFP MCS after 3-day exposure to anticancer treatments (cisplatin, paclitaxel, or two drug formulations developed in-house) and 4-day drug-free growth (Mean ± SD, N = 3-4). (C) Morphological studies of representative A549-iRFP MCS after 1-6 weeks of culturing by contrast phase microscopy (C1, scale bar = 500 µm), fluorescent confocal microscopy (C2), and software simulation (C3). (D) Dynamic change of the iRFP fluorescence, volume, cell viability, and cellular protein of A549-iRFP MCS over 1-6 weeks (N = 10 for iRFP fluorescence and volume; N = 5 for cell viability and cellular protein). (E) Correlation of iRFP fluorescence of A549-iRFP MCS with the other three traditional growth measurements (volume, cell viability, and cellular protein) over 1-6 weeks (N = 60). (F) An example of A549-iRFP MCS of appropriate characteristics for tumor inoculation (scale bar = 500 µm). Abbreviation: MCS = multicellular spheroid. Please click here to view a larger version of this figure.

Figure 3: Tumor progression in the orthotopic murine model of NSCLC. (A) Change in body weight of mice after tumor inoculation (Mean ± SEM, N = 5). (B) Dynamic change of net fluorescent intensity in tumor-bearing mice based on left-side imaging (N = 5). (C) Dynamic change of net fluorescent intensity in tumor-bearing mice based on ventral-side imaging (N = 5). (D) Cancer progression mimicking the four clinical stages of NSCLC. Left-side imaging, ventral-side imaging, anatomical observation (yellow arrows indicate tumors in the lung and on the heart surface), and ex-vivo imaging of representative tumor-bearing mice at four stages. This figure is taken from Huang et al.¹⁸. Abbreviation: NSCLC = non-small cell lung cancer. Please click here to view a larger version of this figure.

Figure 4: Tumor-bearing mice's response to cisplatin. (A) Ventral-side imaging of tumor-bearing mice after treatment with saline or cisplatin (images in red rectangles indicate when the tumor fluorescence can be first visualized in both the left and the right lungs). (B) Change in the body weight of tumor-bearing mice after saline or cisplatin treatments (Mean ± SEM, N = 3-4). (C) Dynamic change of net fluorescent intensity in tumor-bearing mice after saline or cisplatin treatments based on ventral-side imaging (Mean ± SEM, N = 3-4). (D) Percentage of mice showing tumor fluorescence on both the left and the right lungs. (E) Normalized fluorescent intensity of lungs dissected from mice sacrificed at the end of in vivo fluorescent imaging (Mean ± SEM, N = 3-4). Please click here to view a larger version of this figure.

Supplementary Figure S1: 3D cell viability assay on A549-iRFP MCS of various diameters. (A) ~350 µm; (B) ~550 µm; (C) ~750 µm; (D) ~950 µm. Please click here to download this File.

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 standardized the formation of MCS, ensuring their consistency and reliability in our experimental model. This approach enhances the reproducibility of our results and minimizes variability in tumor growth kinetics, thus providing a more robust platform for studying tumor biology and therapeutic interventions.

In contrast to traditional 2D monolayer cell cultures, 3D MCS can grow over a much longer timeframe, at least 6 weeks in this study. To monitor cancer growth and progression, a dynamic, long-term quantitative measurement is highly beneficial. Many existing 3D MCS assays are destructive during the measurements, thus limiting their usefulness for long-term evaluations. The non-destructive nature of measuring the iRFP fluorescent signals allows continual and long-term monitoring of cancer cell growth in 3D MCS. This feature is particularly valuable for assessing the effects of chronic exposure to anticancer agents and for capturing the dynamic evolution of cancer cells over time. The iRFP fluorescent signals emitted by the A549-iRFP 3D MCS displayed a strong correlation with traditional quantitative measurements of spheroid growth, including spheroid volume, cell viability, and cellular protein levels. This highlights the reliability of the fluorescent signals as a proxy for assessing cancer cell growth in MCS. The long wavelength of the iRFP fluorescent signals allows the quantification of cancer growth and progression in small animals, which is critical for the preclinical development of anticancer drugs.

To ensure the consistency of this orthotopic NSCLC model, MCS need to be carefully selected for intrapulmonary inoculation. Similar size and fluorescent intensity of the MCS are necessary to minimize variation; inoculation of larger MCS tends to cause larger variation in tumor progression. Additionally, controlling the roughness of the MCS surface plays a critical role in reducing the variation. While some MCS maintained their smooth edges as they grew larger, others displayed rougher edges as they grew. In our hands, the integral, solid-tumor-like MCS after 2-3 weeks growing in cell culture with 5-10 buddings at the edges yielded more uniform tumor growth in mice, whereas those without rough edges or those with loosened structures and too many buddings yielded more diverse patterns of tumor growth after inoculation.

During the process of MCS transfer, it is essential to handle the 20 G needle with great care. MCS should be kept within the bevel or the metal part of the needle to limit potential damage. The choice of needle size was made deliberately to balance between ensuring the viability of the MCS during inoculation and minimizing the potential damage to mouse lungs. Larger needle sizes could potentially cause damage to the mouse tissues. If MCS become stuck within the needle or if multiple fluorescent dots are observed in the early days following inoculation, it may indicate that the inoculated MCS have been damaged during inoculation. Mice displaying these characteristics were excluded from further studies and classified as unsuccessful inoculation.

The multicellular spheroids (MCS) were inoculated into the left lung rather than the right lung based on anatomical considerations. The left lung has two lobes stacked on top of each other, providing a more consistent and accessible target area compared to the right lung, which has three lobes located side by side. Thus, the left lung allows highly reliable inoculation in a specific area, namely the upper left lobe, which helps reduce variations in tumor origination and progression.

Besides the chest iRFP fluorescence, the body weight of the tumor-bearing mice serves as an important corroborative indicator of tumor burden. As the xenograft cancer progressed after MCS inoculation, the mice first displayed normal behavior, including eating, drinking, and activities when awake, until about Day 40, when the heavy tumor burden, as shown by strong chest iRFP fluorescence, resulted in a noticeable drop in body weight and the loss of the normal behavior. Following humane guidelines, the animals were euthanized at 15-20% loss in body weight. While the timing of the weight loss varied among batches, it matches with the loss of normal animal behavior and thus, can also be used as a reliable indicator of animal health.

The cancer progression in the orthotopic model of NSCLC was primarily monitored through left and ventral side florescent imaging, which showed consistent and steady tumor growth after MCS inoculation. Fluorescence from the ventral side showed a slower increase than the left side probably because of (1) the depth of injection and (2) the presence of both orthotopic and subcutaneous tumors at advanced cancer stages. Specifically, the MCS was inoculated through the rib bones into the thorax, which deposited it on the dorsal surface of the left lung, thus requiring a lag time for the MCS to grow to the ventral surface of the left lung to be imaged. As the tumor grew, it occasionally extended onto the inner surface of the chest wall, thus bringing it closer to the fluorescent scanner to give higher signals for the left-side imaging. Therefore, fluorescent imaging on the ventral side more accurately presented the tumor in the lung but usually took a longer time to detect the tumor. At the end of the in vivo fluorescent imaging, the animals were sacrificed and subjected to open-chest dissection and ex vivo imaging, which not only confirmed the establishment of the orthotopic tumor from the MCS inoculation but also displayed multiple anatomical features that recapitulated the four clinical stages of NSCLC in humans.

In the pilot efficacy study, the strong correlation between the ex vivo imaging of the lungs and the in vivo imaging of tumor-bearing mice corroborates the reliability of the dynamic in vivo fluorescence imaging methodology. The orthotopic model of NSCLC responded reliably to anticancer treatments by both the slower tumor growth as a key indicator of therapeutic efficacy and by the loss of body weight as a key indicator of drug toxicity. Such responses demonstrated the model as a valuable tool for the preclinical assessment and development of anticancer drug candidates. Metastasis, a hallmark of lung cancer, was recapitulated faithfully in the model, and more importantly, was reliably hindered by the anticancer drug cisplatin. Slower tumor growth was observed with only two injections of cisplatin in this pilot study, suggesting that a more significant anticancer effect of cisplatin would result from a more clinically relevant regimen of four weekly doses.

The orthotopic model of NSCLC of this report carries several important advantages over a number of common animal models of lung cancer. Compared to the subcutaneous xenograft models of lung cancer, this model offers a more relevant tissue microenvironment around the tumors; compared to the genetically engineered mouse model, this model offers more convenient experimental procedures and more reproducible tumor progression and animal survival. A comprehensive comparison between this model and other models of lung cancer is available in our previous publication¹⁸. Similar to other xenograft animal models, one limitation of this model is the lack of a complete immune system, which is an important component of human cancers. Additionally, the inoculated MCS in this model occasionally develop the primary xenograft tumor on the inner surface of the chest wall. Therefore, experimental procedures to easily detect and minimize such development should be included in future studies to improve this model. Moreover, studies involving both sexes will be carried out to achieve a more comprehensive understanding of tumor biology and treatment responses in NSCLC. In conclusion, the murine orthotopic lung cancer xenograft model of this report stands as a pioneering and robust tool for recapitulating lung cancer progression and for evaluating anticancer treatments. This model holds immense potential for advancing our understanding of NSCLC pathology and for developing the corresponding therapeutic interventions.

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