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

Our research focused on developing nano-formulations of anti-cancer drugs. In order to evaluate anti-cancer activities, we established mirroring 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 with fluorescent imaging, and respond 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 lacked the fidelity to the tumor microenvironments and disease progression. By utilizing multicellular spheroids and autotrophic inoculation, we bridge the gap, better mimicking non-small cell lung cancer complexity and therapy response.

With this autotrophic animal model, we have opened avenues to investigate non-small cell lung cancer tumor microenvironment and screen normal molecule for anti-cancer activity. Our finding paved the way for deeper explanation into tumor biology and the developments of anti-cancer therapies. 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 microplate using 100 L of complete growth medium supplemented with 0.3%collagen. Centrifuge the microplates at 300 G for 7 minutes at 4 C to facilitate the MCS formation. After centrifugation, examine the spheroid morphology under a microscope to ensure cell aggregation.

Replace 100 L 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 5 to 10 rough buttings and an appropriate diameter. Measure the IRFP fluorescence to select the MCS with fluorescent signals within 1 standard deviation of the average.

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 the 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 1 cm 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 spheroid or MCS from a microplate into a glass Petri dish containing ice cold PBS. Attach a 20-gauge needle to a 100 L glass syringe and pre-cool them on ice. Then draw 20 L of a pre-cooled mixture of PBS and Matrigel.

Use the syringe to quickly aspirate 1 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 3 mm and slowly inject all 20 L 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 nm 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.