establishment of 3d multicellular spheroids from a549-irfp cells for tumor inoculation

Intrapulmonale Inokulation von multizellulären Sphäroiden zur Modellierung des orthotopen NSCLC

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.

Autor im Rampenlicht: Etablierung eines murinen nicht-kleinzelligen Lungenkrebsmodells für die Entwicklung von Nanoformulierungen von Krebsmedikamenten

Konstruktion eines orthotopen Xenotransplantatmodells für nicht-kleinzelligen Lungenkrebs, der das Fortschreiten der Krankheit nachahmt und die Wirkstoffaktivität vorhersagt

Etablierung von multizellulären 3D-Sphäroiden aus A549-iRFP-Zellen für die Tumorinokulation

establishment-3d-multicellular-spheroids-from-a549-irfp-cells-for

Research

JoVE Journal

Cancer Research

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

51 Aufrufe. Etablieren Sie zunächst die dreidimensionalen multizellulären Sphäroide oder MCS aus menschlichen Lungenadenokarzinom- oder A549-iRFP-Zellen. Aussaat der Zellen in einer 96-Well-Mikroplatte mit ultraniedriger Bindung und rundem Boden unter Verwendung von 100 Mikrolitern vollständigem Wachstumsmedium, ergänzt mit 0,3 % Kollagen. Zentrifugieren Sie die Mikrotiterplatten bei 300 g sieben Minuten lang bei vier Grad Celsius, um die MCS-Bildung zu erleichtern....

Serie: Etablierung von multizellulären 3D-Sphäroiden aus A549-iRFP-Zellen für die Tumorinokulation

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.

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