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. 2022 Jan 25;12(4):1002–1021. doi: 10.1158/2159-8290.CD-21-0910

Figure 1.

Figure 1. Fatal brain relapse in the osimertinib-treated, PC9-derived metastatic lung cancer mouse model. A, Schematic representation of the in vivo treatment model derived from the PC9-BrM cell line for metastatic EGFR-mutant lung cancer. Luciferase-labeled human EGFR-mutant PC9-BrM cells were injected into the arterial circulation of immunodeficient mice via intracardiac injection to generate metastases, which were detected by bioluminescence imaging. At 25 days after tumor cell injection, after confirmation of metastatic (met) signal, mice were administered long-term treatment with either vehicle or osimertinib (Osi) at 5 mg/kg body weight/day by oral gavage 5 days per week until the endpoint indicated in B and C, which averaged to 8 months after tumor cell injection. Periods of response to osimertinib and subsequent relapse were detected by bioluminescence imaging. B, Representative images for longitudinal monitoring of metastatic progression with vehicle (Veh) or osimertinib treatment by weekly bioluminescence imaging, with progressive development of osimertinib-refractory brain relapse in mice. Vehicle-treated mice developed bone, brain, and lymph node metastases and were euthanized when weight loss was >20% or when the body-conditioning score (BCS) reached 2. Osimertinib-treated mice were monitored for the emergence and progression of osimertinib-refractory metastasis in the brain and euthanized when either weight loss was >20%, the BCS reached 2, or mice developed paralysis or seizure-like symptoms due to brain metastasis. Days represent days after initial tumor cell injection. Photon-flux scales are indicated below the images. C, Kaplan–Meier plot for brain metastasis PFS of mice from the experiment described in A and B. Data were analyzed using the log-rank test: χ2  =  19.33, degrees of freedom (d.f.)  =  1, P < 0.0001, n = 10 for vehicle-treated mice and 10 for osimertinib-treated mice. D, Schematic representation of the experimental design to derive osimertinib treatment–refractory Tr-BrM cells from relapsed brain metastases from the mice described in A–C that were injected with PC9-BrM cells and treated long-term with osimertinib. E, Kaplan–Meier plot for brain metastasis PFS of mice injected with PC9-Tr-BrM cells followed by treatment with either vehicle or osimertinib. Data were analyzed using the log-rank test: χ2  =  1.325, d.f.  =  1, P value not significant (ns), n = 20 for vehicle-treated mice and 17 for osimertinib-treated mice. F, Representative images of human CK7 IHC on brain sections from mice injected with either PC9-BrM cells (top) or PC9-Tr-BrM cells (bottom) and treated with either vehicle (left) or osimertinib (right). Mice were euthanized at 7 weeks after tumor cell injection. Scale bars, 200 μm. G, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis that are represented in F. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 10 for vehicle-treated mice bearing PC9-BrM or PC9-Tr-BrM metastases and n = 11 for osimertinib-treated mice bearing PC9-BrM or PC9-Tr-BrM metastases.

Fatal brain relapse in the osimertinib-treated, PC9-derived metastatic lung cancer mouse model. A, Schematic representation of the in vivo treatment model derived from the PC9-BrM cell line for metastatic EGFR-mutant lung cancer. Luciferase-labeled human EGFR-mutant PC9-BrM cells were injected into the arterial circulation of immunodeficient mice via intracardiac injection to generate metastases, which were detected by bioluminescence imaging. At 25 days after tumor cell injection, after confirmation of metastatic (met) signal, mice were administered long-term treatment with either vehicle or osimertinib (Osi) at 5 mg/kg body weight/day by oral gavage 5 days per week until the endpoint indicated in B and C, which averaged to 8 months after tumor cell injection. Periods of response to osimertinib and subsequent relapse were detected by bioluminescence imaging. B, Representative images for longitudinal monitoring of metastatic progression with vehicle (Veh) or osimertinib treatment by weekly bioluminescence imaging, with progressive development of osimertinib-refractory brain relapse in mice. Vehicle-treated mice developed bone, brain, and lymph node metastases and were euthanized when weight loss was >20% or when the body-conditioning score (BCS) reached 2. Osimertinib-treated mice were monitored for the emergence and progression of osimertinib-refractory metastasis in the brain and euthanized when either weight loss was >20%, the BCS reached 2, or mice developed paralysis or seizure-like symptoms due to brain metastasis. Days represent days after initial tumor cell injection. Photon-flux scales are indicated below the images. C, Kaplan–Meier plot for brain metastasis PFS of mice from the experiment described in A and B. Data were analyzed using the log-rank test: χ2  =  19.33, degrees of freedom (d.f.)  =  1, P < 0.0001, n = 10 for vehicle-treated mice and 10 for osimertinib-treated mice. D, Schematic representation of the experimental design to derive osimertinib treatment–refractory Tr-BrM cells from relapsed brain metastases from the mice described in A–C that were injected with PC9-BrM cells and treated long-term with osimertinib. E, Kaplan–Meier plot for brain metastasis PFS of mice injected with PC9-Tr-BrM cells followed by treatment with either vehicle or osimertinib. Data were analyzed using the log-rank test: χ2  =  1.325, d.f.  =  1, P value not significant (ns), n = 20 for vehicle-treated mice and 17 for osimertinib-treated mice. F, Representative images of human CK7 IHC on brain sections from mice injected with either PC9-BrM cells (top) or PC9-Tr-BrM cells (bottom) and treated with either vehicle (left) or osimertinib (right). Mice were euthanized at 7 weeks after tumor cell injection. Scale bars, 200 μm. G, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis that are represented in F. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 10 for vehicle-treated mice bearing PC9-BrM or PC9-Tr-BrM metastases and n = 11 for osimertinib-treated mice bearing PC9-BrM or PC9-Tr-BrM metastases.