Name: Författare i fokus: Etablering av en murin icke-småcellig lungcancermodell för att utveckla nanoformuleringar av anticancerläkemedel
Uploaded: 2024-05-10T14:50:00
Description: 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

Detta arbete stöddes av SAAG- och SEED-anslag från University of the Pacific. Vi tackar Dr. William Chan för att ha beviljat åtkomst till Odyssey Infrared Imaging 205 System och Dr. John Livesey för att ha beviljat tillgång till SpectraMax iD3 plattläsare. Vi tackar Dr. Melanie Felmlee för de tekniska råden om djurprotokollen.

Djurstudien utfördes med godkännande av Institutional Animal Care and Use Committee (IACUC) vid University of the Pacific (Animal Protocols 19R10 och 22R10). Åtta hanar av athymiska nakna möss i åldern 5-6 veckor, som väger 20-25 g, avlade med den refererade gnagardieten och inhysta under patogenfria (SPF) förhållanden, användes för den aktuella studien. Burar, sängkläder och dricksvatten autoklaverades och byttes regelbundet. En schematisk bild av tumörinokulering hos möss visas i figu...

Intrapulmonell inokulering av flercelliga sfäroider för att modellera ortotopisk 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+pathologic+classification+of+lung+cancer:+Relevance+for+clinical+practice+and+clinical+trials.">New pathologic classification of lung cancer: Relevance for clinical practice and clinical trials.</a> J Clin Oncol. 31 (8), 992-1001 (2013).</li><li>Ducote, T. J., 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=Using+artificial+intelligence+to+identify+tumor+microenvironment+heterogeneity+in+non-small+cell+lung+cancers.">Using artificial intelligence to identify tumor microenvironment heterogeneity in non-small cell lung cancers.</a> Lab Invest. 103 (8), 100176 (2023).</li><li>Lim, Z. F., Ma, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+insights+of+tumor+heterogeneity+and+drug+resistance+mechanisms+in+lung+cancer+targeted+therapy.">Emerging insights of tumor heterogeneity and drug resistance mechanisms in lung cancer targeted therapy.</a> J Hematol Oncol. 12 (1), 134 (2019).</li><li>Wang, D. C., Wang, W., Zhu, B., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+heterogeneity+and+new+strategies+for+drug+therapy.">Lung cancer heterogeneity and new strategies for drug therapy.</a> Annu Rev Pharmacol Toxicol. 58, 531-546 (2018).</li><li>Peterson, J. K., Houghton, P. 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. D., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+for+use+of+tumor+spheroids+as+models+to+test+drug+delivery+and+efficacy.">Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.</a> J Control Release. 164 (2), 192-204 (2012).</li><li>Huang, B. W., Gao, J. 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. U., Yang, Y. W., Buchanan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer:+Biology+and+treatment+options.">Lung cancer: Biology and treatment options.</a> Biochim Biophys Acta. 1856 (2), 189-210 (2015).</li><li>Detterbeck, F. C., Boffa, D. J., Kim, A. W., Tanoue, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eighth+edition+lung+cancer+stage+classification.">The eighth edition lung cancer stage classification.</a> Chest. 151 (1), 193-203 (2017).</li></ol>

Konstruktionen av A549-iRFP MCS är en okomplicerad och mycket reproducerbar laboratorieprocedur och kan översättas till MCS-bildning för flera cellinjer. MCS som genereras med hjälp av centrifugering och kollagen uppvisar en mer integrerad och solid-tumörliknande struktur inom 3-4 dagar. Denna metod säkerställer bildandet av robusta sfäroider som behåller sin integrerade struktur under längre perioder, vanligtvis 2-3 veckor eller ännu längre tills små knoppar börjar dyka upp. Genom att använda centrifuger...

Bland alla onkologiska sjukdomar orsakar lungcancer inte bara den högsta förlusten av liv utan kräver också det näst högsta antalet nya patienter varje år iUSA1. Denna förödande malignitet står som ett stort hinder i modern sjukvård, vilket uppmanar till en djupare förståelse av dess intrikata biologi och mer effektiva terapeutiska modaliteter2. Icke-småcellig lungcancer (NSCLC) står för 85 % av lungcancern och tenderar att utvecklas till solida tumörer3. En av de främsta utmaningarna inom lungcancer är den dynamiska och heterogena mikromiljön i tumören, som i hög grad påverkar cancerns progression och svar på terapeutiska interventioner 4,5,6. En djupare förståelse av samspelet mellan cancerceller och deras mikromiljö i olika stadier av NSCLC kräver förfinade patologiska modeller som rekapitulerar de histologiska egenskaperna hos NSCLC-progression.
I detta avseende framstår ortotopiska djurmodeller som en lovande väg för NSCLC-forskning. Till skillnad från vanliga subkutana xenograftmodeller7, har ortotopiska modeller cancerceller som inokuleras direkt i ursprungsorganet. För lungcancer innebär detta att man implanterar cancerceller direkt i lungvävnaden 8,9. Följaktligen efterliknar ortotopiska modeller av lungcancer bättre den ursprungliga tumörmikromiljön, inklusive de närliggande vävnaderna, kärlen och immunkomponenterna, vilket förbättrar deras fysiologiska och kliniska relevans.
Tredimensionella flercelliga sfäroider (MCS) representerar ett annat lovande tillvägagångssätt för att rekapitulera egenskaper hos tumörmiljön. De flesta cancerformer kännetecknas av sin komplexa tumörmikromiljö, inklusive de olika cell-cellinteraktionerna, den extracellulära matrisen och gradienterna i syre och näringsämnen10,11. Traditionella 2D-cellkulturer saknar den rumsliga och strukturella komplexiteten för att rekapitulera dessa tumörspecifika egenskaper12. Däremot har MCS av lämplig storlek en heterogen struktur med en hypoxisk och nekrotisk kärna, som rekapitulerar inte bara den intratumorala mikromiljön utan också den fysiologiska barriären mot läkemedelspenetration, vilket är en viktig mekanism för läkemedelsresistens vid anticancerbehandling 13,14,15.
Genom att dra nytta av både de ortotopiska djurmodellerna och MCS-odlingsteknikerna har MCS inokulerats på immunsupprimerade möss för att framgångsrikt konstruera ortotopiska modeller av bröstcancer och prostatacancer16,17. Här redogör vi för den detaljerade metodiken för att konstruera och karakterisera en murin ortotopisk xenograftmodell av lungcancer. Denna metod använder intrapulmonell inokulering av 3D MCS härledd från fluorescerande humana lungcancerceller (A549-iRFP)18. Denna modell erbjuder en exceptionell möjlighet att observera in vivo-progressionen av lungcancer genom stadier som är nära parallella med de fyra kliniska stadierna av NSCLC. Dessutom svarade xenograftcancern i denna modell på det kliniskt etablerade läkemedlet mot lungcancer, cisplatin.

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			<td>100 &#181;L Glass Syringe</td>
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			<td>Thermo Fisher Scientific Inc.</td>
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			<td>96-well Ultra-Low-Attachment Spheroid Microplate&#160;</td>
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			<td>Charles River Laboratories, Inc.</td>
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			<td>NDC Number: 42023-179-05</td>
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			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
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			<td>DMEM</td>
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			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cytiva HyClone</td>
			<td>SH3039603</td>
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			<td>ImageJ</td>
			<td>Open source tool (https://imagej.net/ij/)</td>
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			<td>Ketamine</td>
			<td>Patterson Veterinary</td>
			<td>NDC Number: 50989-0161-06</td>
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			<td>Microscope&#160;</td>
			<td>Keyence</td>
			<td>Model number: BZ-X710</td>
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			<td>Pearl Trilogy small animal imaging system&#160;</td>
			<td>LI-COR</td>
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			<td>Penicillin-Streptomycin&#160;</td>
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			<td>MT30002CI</td>
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			<td>Puromycin</td>
			<td>Thermo Fisher Scientific Inc.</td>
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			<td>ReViSP software from MATLAB&#160;</td>
			<td>Open source tool on Sourceforge (https://sourceforge.net/projects/revisp/)</td>
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			<td>Surgical Clips--AutoClip System</td>
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			<td>Patterson Veterinary</td>
			<td>NDC Number: 61133-6017-01</td>
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construction of an orthotopic xenograft model of non-small cell lung cancer mimicking disease progression and predicting drug activities

Icke-småcellig lungcancer (NSCLC) är en mycket dödlig sjukdom med en komplex och heterogen tumörmikromiljö. För närvarande finns det vanliga djurmodeller baserade på subkutan inokulering av cancercellssuspensioner som inte rekapitulerar tumörens mikromiljö i NSCLC. Här beskriver vi en murin ortotopisk xenograftmodell för lungcancer som använder intrapulmonell inokulering av tredimensionella flercelliga sfäroider (MCS). Specifikt odlades fluorescerande humana NSCLC-celler (A549-iRFP) i lågfästa 96-brunns mikroplattor med kollagen i 3 veckor för att bilda MCS, som sedan inokulerades interkostalt i den vänstra lungan hos atymiska nakna möss för att etablera den ortotopiska lungcancermodellen.
Jämfört med den ursprungliga A549-cellinjen svarade MCS i A549-iRFP-cellinjen på liknande sätt på anticancerläkemedel. Den fluorescerande signalen med lång våglängd från A549-iRFP-cellerna korrelerade starkt med vanliga markörer för cancercellstillväxt, inklusive sfäroidvolym, cellviabilitet och cellulär proteinnivå, vilket möjliggör dynamisk övervakning av cancertillväxten in vivo genom fluorescensavbildning. Efter inokulering i möss utvecklades A549-iRFP MCS-xenograften på ett tillförlitligt sätt genom faser som mycket liknade de kliniska stadierna av NSCLC, inklusive expansionen av den primära tumören, uppkomsten av närliggande sekundära tumörer och metastaser av cancerceller till den kontralaterala högra lungan och avlägsna organ. Dessutom svarade modellen på det ledande läkemedlet mot lungcancer, cisplatin, med den förväntade toxiciteten och långsammare cancerprogression. Därför skulle denna murina ortotopiska xenograftmodell av NSCLC fungera som en plattform för att rekapitulera sjukdomens progression och underlätta utvecklingen av potentiella anticancerläkemedel.

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.

Författare i fokus: Etablering av en murin icke-småcellig lungcancermodell för att utveckla nanoformuleringar av anticancerläkemedel

Konstruktion av en ortotopisk xenograftmodell av icke-småcellig lungcancer som efterliknar sjukdomsprogression och förutsäger läkemedelsaktiviteter

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.

Karakterisering av A549-iRFP MCS A549-iRFP MCS odlades framgångsrikt i sfäroidmikroplattor med hjälp av kollagen och centrifugering. När MCS nådde en diameter på cirka 500 μm efter 1 vecka, exponerades både A549 och A549-iRFP MCS för en mängd olika anticancerläkemedel och formuleringar under 3 dagar och bibehölls sedan i läkemedelsfritt odlingsmedium i ytterligare 4 dagar. A549-iRFP MCS uppvisade ett responsmönster som nära speglade det hos de ursprungliga A549-cellerna. A549 och A549-i...

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

Denna studie presenterar en ortotopisk modell för icke-småcellig lungcancer (NSCLC) baserad på intrapulmonell inokulering av flercelliga sfäroider av fluorescerande A549-iRFP-celler. Modellen rekapitulerar kliniska NSCLC-stadier och svarar på cisplatin, enligt dynamisk in vivo-övervakning av långvågig fluorescens.

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

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Cancer Research

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.

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.

Establishment of 3D Multicellular Spheroids from A549-iRFP Cells for Tumor Inoculation

To begin, establish the three dimensional multicellular spheroids or MCS from human lung adenocarcinoma or A549 iRFP cells. Seed the cells in 96 well ultra-low attachment round bottom spheroid micro plate using 100 microliters of complete growth medium supplemented with 0.3%collagen. Centrifuge the micro plates at 300 G for seven minutes at four degrees Celsius to facilitate the MCS formation.After centrifugation, examine the spheroid morphology under a microscope to ensure cell aggregation. Replace 100 microliters of growth medium in each well with fresh complete growth medium every other day. While observing under a microscope, choose spheroids with a round shape and overall smooth edges, but with five to 10 rough buddings and an appropriate diameter.Measure the iRFP fluorescence to select the MCS with fluorescent signals within one standard deviation of the average.

Etablering av 3D multicellulära sfäroider från A549-iRFP-celler för tumörinokulering

Mouse Surgery for Intrapulmonary Inoculation of A549-iRFP Cell-Derived Multicellular Spheroids

Begin the surgical procedure on the properly anesthetized mouse by injecting buprenorphine hydrochloride subcutaneously to reduce the pain. Apply ophthalmic ointment to the eyes to prevent dryness. Place the mouse in a dorsal posture and secure the limbs in a stretching position with tapes.Disinfect the back skin using alternating swabs of iodine and alcohol, at least three times each. Next, using surgical scissors, cut a 0.5-to-one-centimeter incision on the left back side of the animal. Carefully use forceps to separate the muscle and fat tissues until the chest wall and lung motion are visible.Using a pipette, transfer one selected multicellular steroid, or MCS, from a microplate into a glass Petri dish containing ice-cold PBS. Attach a 20-gauge needle to a 100-microliter glass syringe and pre-cool them on ice. Then draw 20 microliters of a pre-cooled mixture of PBS and Matrigel.Use the syringe to quickly aspirate one MCS from the Petri dish in a minimum volume of PBS-Matrigel mixture, keeping the spheroid in the metal part of the needle. Gently insert the needle vertically between two rib bones to a depth of approximately three millimeters and slowly inject all 20 microliters of the mixture containing the aspirated MCS. Then carefully remove the needle.Apply triple antibiotic ointment to the wound. Seal the incision with surgical clips to be removed after two weeks. 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 to 60 minutes until it wakes up and moves properly. Using a small animal imaging system at a 700-nanometer channel, measure the IRFP fluorescent signal from the tumor xenograft of the anesthetized mouse every three to four days. Measure the fluorescence in four postures, left, right, dorsal and ventral.The net fluorescence intensity from both the left and ventral sides showed a similar trend of tumor progression, with fluorescence from the ventral side growing slightly slower than the fluorescence from the left side.