Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells

Abstract

Background: Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC.

Methods: Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models.

Results: Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours.

Conclusions: Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

Figure 1. Study flowchart.

a. A549 cells stably transfected with luciferase were tested by direct application of serum saline (left side, control) or luciferine (right side), and analysed after optical imaging (Time 1 min after addition of luciferine, exposure 1 min, bin medium). b. Animals were divided in 4 groups, subcutaneous injection (group 1-SC), percutaneous orthotopic injection under radiographic control (group 2-POI), surgical orthotopic implantation after orotracheal intubation and thoracotomy approach (group 3-SOI), and transpleural orthotopic injection after costal layer surgical exposure (group 4-TOI).

Figure 2. Follow-up using bioluminescent imaging and survival curves.

a. Evolution of the photons count (expressed as 10⁶ photons/s) over time, from day 0 (implantation) to day 63 (end of the bioluminescent follow up). For each time, the mean and standard deviation of the animals alive are reported. In group 2-POI, percutaneous injection led to pleural seeding in 53% of the cases, as postoperative X-ray showed contrast agent in either left pleural space, right pleural space (anatomical continuum), or both. b. Representative evolution of the bioluminescent signal, from day 14 to day 42. In group 2-POI, pleural seeding created a signal located at the right basis of the thorax, contradicting with the primary goal of this study. No subsequent follow-up as been performed for this group. c. Survival curve from the time of tumor implantation to 2 months using the Kaplan-Meier method and 95% confidence interval bars. The 2-month survival is 100% in group 1-SC, 40% in group 3-SOI, and 65% in group 4-TOI.

Figure 3. Pathological examination.

Surgical orthotopic implantation (Group 3) resulted in localized infiltrating tumors, whereas percutaneous orthotopic injection (Group 2) and transpleural orthotopic injection (group 4) resulted in localized rounded nodules. Histological examination revealed a high proportion of undifferentiated carcinoma in group 1-SC tumor, but a low proportion in the 3 experimental groups. Furthermore, the 3 experimental groups were associated with more capsule rupture and vascular emboli than group 1-SC.

Figure 4. Identification of circulating tumor cells.

a. Representation of the staining of circulating cells using the Cellsearch® assay. CTC are defined as CD45−, DAPI+, and CKPE+ cells. - Upper line: as a positive control, 50 and 500 A549luc cells were analysed in a solution containing 600 µL of medium and 7 mL of healthy human blood. Here, as an example, both CD45−, DAPI+, CKPE+ cells are tumor cells. - Bottom line: representation of the results of the experimental groups. Here, as an example, this CD45−, DAPI+, CKPE+ cell is a CTC. b. Correlation between the Cellsearch ® CTC count and the number of tumor cells in solution. As controls, 0 (negative control), 50 and 500 (positive controls) A549luc cells were analysed in a solution containing 600 µL of medium and 7 mL of healthy human blood. This experiment demonstrated a correlation between the Cellsearch® CTC count and the number of tumor cells in solution (R² = .99857). c. Number of CTC according to the experimental group, CTC group 1 (SC) or CTC group 2 (orthotopic). The overall positivity of the Cellsearch ® assay is comparable in both groups (83%), with a trend toward more CTC in the orthotopic rather than in the subcutaneous model. d. Representation of the bioluminescent signal and number of CTC detected in each of the 12 animals.