This work was supported by SAAG and SEED grants from the University of the Pacific. We thank Dr. William Chan for granting access to the Odyssey Infrared Imaging 205 System and Dr. John Livesey for granting access to the SpectraMax iD3 plate reader. We thank Dr. Melanie Felmlee for the technical advice on the animal protocols.

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. ...

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 70 (1), 7-30 (2020).</li><li>Bray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2018:+Globocan+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA Cancer J Clin. 68 (6), 394-424 (2018).</li><li>Travis, W. D., Brambilla, E., Riely, G. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+pharmacology+and+in+vivo+cancer+models+in+preclinical+and+clinical+drug+development.">Integrating pharmacology and in vivo cancer models in preclinical and clinical drug development.</a> Eur J Cancer. 40 (6), 837-844 (2004).</li><li>Madero-Visbal, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+correlates+with+tumor+progression+in+an+orthotopic+mouse+model+of+lung+cancer.">Bioluminescence imaging correlates with tumor progression in an orthotopic mouse model of lung cancer.</a> Surg Oncol. 21 (1), 23-29 (2012).</li><li>Mclemore, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+intrapulmonary+model+for+orthotopic+propagation+of+human+lung+cancers+in+athymic+nude+mice.">Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice.</a> Cancer Res. 47 (19), 5132-5140 (1987).</li><li>Zanoni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3d+tumor+spheroid+models+for+in+vitro+therapeutic+screening:+A+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3d tumor spheroid models for in vitro therapeutic screening: A systematic approach to enhance the biological relevance of data obtained.</a> Sci Rep. 6, 19103 (2016).</li><li>Kim, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Semin Cancer Biol. 15 (5), 365-377 (2005).</li><li>Chaicharoenaudomrung, N., Kunhorm, P., Noisa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+systems+as+an+in+vitro+platform+for+cancer+and+stem+cell+modeling.">Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling.</a> World J Stem Cells. 11 (12), 1065-1083 (2019).</li><li>Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. 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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+3d+cultured+multicellular+spheroid+tumor+models+in+tumor-targeted+drug+delivery+system+research.">Application of 3d cultured multicellular spheroid tumor models in tumor-targeted drug delivery system research.</a> J Control Release. 270, 246-259 (2018).</li><li>Hirschhaeuser, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+An+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: An underestimated tool is catching up again.</a> J Biotechnol. 148 (1), 3-15 (2010).</li><li>Sethi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3d+tumor+tissue+analogs+and+their+orthotopic+implants+for+understanding+tumor-targeting+of+microenvironment-responsive+nanosized+chemotherapy+and+radiation.">3d tumor tissue analogs and their orthotopic implants for understanding tumor-targeting of microenvironment-responsive nanosized chemotherapy and radiation.</a> Nanomedicine. 11 (8), 2013-2023 (2015).</li><li>Valta, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+of+lucap+136+patient-derived+xenograft+enables+versatile+preclinical+models+of+prostate+cancer.">Spheroid culture of lucap 136 patient-derived xenograft enables versatile preclinical models of prostate cancer.</a> Clin Exp Metastasis. 33 (4), 325-337 (2016).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapulmonary+inoculation+of+multicellular+spheroids+to+construct+an+orthotopic+lung+cancer+xenograft+model+that+mimics+four+clinical+stages+of+non-small+cell+lung+cancer.">Intrapulmonary inoculation of multicellular spheroids to construct an orthotopic lung cancer xenograft model that mimics four clinical stages of non-small cell lung cancer.</a> J Pharmacol Toxicol Methods. , 106885 (2020).</li><li>Lemjabbar-Alaoui, H., Hassan, O. 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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...

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&#39;s progression and responses to therapeutic interventions4,5,6. 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 tissue8,9. 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 nutrients10,11. 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 therapy13,14,15.
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 cancer16,17. 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.

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			<td>100 &#181;L Glass Syringe</td>
			<td>Hamilton</td>
			<td>Part/REF #80601</td>
			<td></td>
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			<td>20 G Needle</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>14 826D</td>
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			<td>96-well Ultra-Low-Attachment Spheroid Microplate&#160;</td>
			<td>Corning</td>
			<td>15-100-173</td>
			<td></td>
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		<tr>
			<td>A549-iRFP</td>
			<td>Imanis Life Sciences&#160;</td>
			<td>CL082-STAN</td>
			<td></td>
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			<td>AIN-93M Mature Rodent Diet&#160;</td>
			<td>Research Diets, Inc.</td>
			<td>D10012M</td>
			<td></td>
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			<td>Athymic Nude Mouse</td>
			<td>Charles River Laboratories, Inc.</td>
			<td>Strain Code: 490; homozygous</td>
			<td></td>
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			<td>BCA</td>
			<td>Pierce</td>
			<td>23227</td>
			<td></td>
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			<td>Buprenorphine Hydrochloride</td>
			<td>Patterson Veterinary</td>
			<td>NDC Number: 42023-179-05</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9683</td>
			<td></td>
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		<tr>
			<td>Collagen</td>
			<td>Gibco</td>
			<td>A1064401&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>MT10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cytiva HyClone</td>
			<td>SH3039603</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Open source tool (https://imagej.net/ij/)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Studio&#160;</td>
			<td>LI-COR</td>
			<td>Version 5.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>NDC Number: 17033-0091-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Patterson Veterinary</td>
			<td>NDC Number: 50989-0161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Keyence</td>
			<td>Model number: BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Infrared Imaging 205 System&#160;</td>
			<td>LI-COR</td>
			<td>Model number: 9140</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pearl Trilogy small animal imaging system&#160;</td>
			<td>LI-COR</td>
			<td>Model number: 9430</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Corning</td>
			<td>MT30002CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>AAJ67236XF</td>
			<td></td>
		</tr>
		<tr>
			<td>ReViSP software from MATLAB&#160;</td>
			<td>Open source tool on Sourceforge (https://sourceforge.net/projects/revisp/)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Clips--AutoClip System</td>
			<td>Fine Science Tools</td>
			<td>12020-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Patterson Veterinary</td>
			<td>NDC Number: 61133-6017-01</td>
			<td></td>
		</tr>
	</tbody>
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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

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

Our research focused on developing nanoformulations of anticancer drugs. In order to evaluate anticancer activities, we established this murine non-small cell lung cancer model by inoculation of multicellular spheroids. We aim to validate this model's ability to replicate clinical stages, drug tumor progression, reflux imaging, and response to therapeutic treatments.We aim to establish a more clinically relevant model of non-small cell lung cancer. Traditional model using subcutaneous inoculation of 2D cell suspension lack the fidelity to a tumor microenvironment and disease progression. By utilizing multicellular spheroids and orthotopic inoculation, we bridge this gap, better mimicking non-small cell lung cancer complexity and therapy response.With this orthotopic animal model, we have opened avenues to investigate non-small cell lung cancer tumor microenvironment and screen normal molecule for anticancer activity. Our finding paved the way for deeper exploration into tumor biology and the developments of anticancer therapies.

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 &#181;m 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...

Read this Scientific Article Protocol about Construction of An Orthotopic Xenograft Model of Non-small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities at JoVE.com

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 ...