Name: construction of an orthotopic xenograft model of non-small cell lung cancer mimicking disease progression and predicting drug activities
Uploaded: 2024-05-10T14:50:00.000+00:00
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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

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. 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
<ol>
	<li>Incubate A549-iRFP cells (human lung adenocarcinoma) in a humidified atmosphere with 5% CO2 at 37 &#176;C and grow them in complete growth medium DMEM containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1 &#181;g/mL puromycin.</li>
	<li>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 &#181;L of complete growth medium supplemented with 0.3% collagen.</li>
	<li>Centrifuge the microplates at 300 &#215; g for 7 min at 4 &#176;C to facilitate the MCS formation. Examine the MCS morphology under a microscope to ensure cell aggregation after centrifugation.</li>
	<li>After 48 h, add 100 &#181;L of complete growth medium to each well to achieve a total volume of 200 &#181;L of growth medium per well.</li>
	<li>Replace 100 &#181;L of growth medium in each well with fresh complete growth medium every other day.</li>
</ol>
2. Characterization of A549-iRFP MCS
<ol>
	<li>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.
	<ol>
		<li>Open one MCS image in ImageJ software.</li>
		<li>Use Freehand selections to select the edge of the MCS and add it to ROI Manager.</li>
		<li>Click Edit | Selection | Create Mask.</li>
		<li>Click Edit | Invert.</li>
		<li>Click File | Save As | Tiff.</li>
		<li>Open ReViSP software.</li>
		<li>Open the saved TIFF image by clicking Browse.</li>
		<li>Click Start to complete the MCS simulation.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Open Image Studio software.</li>
		<li>In the Channels tab, select 700 and Intensity = 5.</li>
		<li>In the Scan Controls tab, select 84 &#181;m, Medium, 0.0 mm, and Flip Image. Keep everything else as default.</li>
		<li>Click Start to start scanning.</li>
	</ol>
	</li>
	<li>Assess and quantify cell viability using the 3D cell viability assay according to the vendor protocol.</li>
	<li>Assess and quantify cellular protein using the BCA Assay according to the vendor protocol.</li>
</ol>
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.
<ol>
	<li>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 &#181;m.</li>
	<li>Measure the iRFP fluorescence (refer to step 2.2) and select the MCS whose fluorescent signals are within one standard deviation of the average.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Inject 0.05 mg/kg of buprenorphine hydrochloride subcutaneously to reduce the pain.</li>
	<li>Apply ophthalmic ointment on the eyes to prevent dryness.</li>
	<li>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.</li>
	<li>Disinfect the back skin by swabbing with alternating rounds of iodine and alcohol, at least three times each.</li>
	<li>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.</li>
	<li>Transfer one selected MCS from a microplate into a glass Petri dish containing ice-cold PBS, using a pipette.</li>
	<li>Attach a 20 G needle onto a 100 &#181;L glass syringe, precool them on ice, and draw 20 &#181;L of a precooled mixture of PBS and Matrigel (1:1 v/v).</li>
	<li>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.</li>
	<li>Gently insert the needle vertically between two rib bones to ~3 mm depth and slowly inject all the 20 &#181;L PBS and Matrigel mixture that contains the MCS. 
	NOTE: Avoid excessive force during the insertion to prevent damage to the lung, vasculature, or heart.</li>
	<li>Carefully remove the needle and apply triple antibiotic ointment to the wound.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Inject 0.05 mg/kg of buprenorphine hydrochloride subcutaneously every 8 h for the first 48 h to reduce the pain.</li>
</ol>
5. Postsurgical monitoring
<ol>
	<li>Measure the body weight every 3-4 days.</li>
	<li>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.</li>
	<li>Quantify the net fluorescent intensity of the xenograft cancer in vivo using software (e.g., Image Studio).
	<ol>
		<li>Use a fixed rectangle (A1, 200 x 125 pixels) as the sampling window for the chest area of the mice.</li>
		<li>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.</li>
		<li>Use another smaller rectangle (A2,&#160;25 x 40 pixels) to measure the background fluorescence (F2) of the mice in the thigh area.</li>
		<li>Use equation (1) to calculate the net fluorescent intensity of the xenograft cancer in each mouse: 
		<img alt="Equation 1" src="/files/ftp_upload/66787/66787eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1)</li>
	</ol>
	</li>
</ol>

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

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The authors have no conflicts of interest to declare.

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.

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.

<table><tbody><tr><td>100 &amp;micro;L Glass Syringe</td><td>Hamilton</td><td>Part/REF #80601</td><td></td></tr><tr><td>20 G Needle</td><td>Thermo Fisher Scientific Inc.</td><td>14 826D</td><td></td></tr><tr><td>96-well Ultra-Low-Attachment Spheroid Microplate&amp;nbsp;</td><td>Corning</td><td>15-100-173</td><td></td></tr><tr><td>A549-iRFP</td><td>Imanis Life Sciences&amp;nbsp;</td><td>CL082-STAN</td><td></td></tr><tr><td>AIN-93M Mature Rodent Diet&amp;nbsp;</td><td>Research Diets, Inc.</td><td>D10012M</td><td></td></tr><tr><td>Athymic Nude Mouse</td><td>Charles River Laboratories, Inc.</td><td>Strain Code: 490; homozygous</td><td></td></tr><tr><td>BCA</td><td>Pierce</td><td>23227</td><td></td></tr><tr><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></tr><tr><td>Collagen</td><td>Gibco</td><td>A1064401&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>LI-COR</td><td>Model number: 9430</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</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&amp;nbsp;</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></table>

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.

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-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 &#181;m 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 &#60; 0.0001), cell viability (Pearson r = 0.7666, p &#60; 0.0001), and cellular protein (Pearson r = 0.7317, p &#60; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66787/66787fig01.jpg" /> 
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&#39;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66787/66787fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66787/66787fig02.jpg" /> 
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 &#177; 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 &#177; 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 &#181;m), 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 &#181;m). Abbreviation: MCS = multicellular spheroid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66787/66787fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66787/66787fig03.jpg" /> 
Figure 3: Tumor progression in the orthotopic murine model of NSCLC. (A) Change in body weight of mice after tumor inoculation (Mean &#177; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66787/66787fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66787/66787fig04.jpg" /> 
Figure 4: Tumor-bearing mice&#39;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 &#177; 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 &#177; 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 &#177; SEM, N = 3-4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66787/66787fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: 3D cell viability assay on A549-iRFP MCS of various diameters. (A) ~350 &#181;m; (B) ~550 &#181;m; (C) ~750 &#181;m; (D) ~950 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66787/66787_Supplementary Figure S1.zip" target="_blank">Please click here to download this File.</a>

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

Author Spotlight: Establishing a Murine Non-Small Cell Lung Cancer Model for Developing Nanoformulations of 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.

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 ...

construction-an-orthotopic-xenograft-model-non-small-cell-lung-cancer

Nanyang Technological University

Research

JoVE Journal

Cancer Research

872 Views.  University of the Pacific. Ons onderzoek richtte zich op de ontwikkeling van nanoformuleringen van geneesmiddelen tegen kanker. Om de antikankeractiviteiten te evalueren, hebben we dit niet-kleincellige longkankermodel bij muizen opgesteld door inoculatie van meercellige sferoïden. We streven ernaar het vermogen van dit model te valideren om klinische stadia, progressie van geneesmiddeltumoren, refluxbeeldvorming en respons op therapeutische behandelingen te repliceren.We strev...

Video: Auteur in de schijnwerpers: Vaststelling van een niet-kleincellig longkankermodel voor muizen voor het ontwikkelen van nanoformuleringen van geneesmiddelen tegen kanker

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioengineering

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Medicine

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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

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