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

Figure 3.

Figure 3. S100A9-proficient cells promote postcolonization growth in the brain. A, Schematic representation of the experimental design to quantify seeding in the brain. PC9-Tr-BrM cells expressing either lenti-control (Lenti-Con) or S100A9i were injected into the arterial circulation of immunodeficient mice via intracardiac injection. Seven days later, brains were isolated, sectioned, and analyzed by IHC for human CK7. CK7-immunostained cancer cells were then counted to compare seeding of cancer cells in the brain parenchyma between experimental groups. B, Quantitative analysis of the experiment described in A. Tumor cells were counted in 10 sections of 20 μm each per brain. Data are presented as mean values ± SEM. The P value was determined by a two--tailed, unpaired Mann–Whitney test. N = 4 for Lenti-Con; n = 5 for S100A9i. ns, P value not significant. C, Schematic representation of the experimental design to analyze postcolonization growth in the brain. PC9-Tr-BrM cells expressing either Lenti-Con or S100A9i were injected into the arterial circulation of immunodeficient mice via intracardiac injection. At 7 weeks after injection, brain tissues were collected, and sections were analyzed by immunostaining for phospho-histone H3 (Ser10) to compare the number of mitotically active cancer cells between the experimental groups. D, Representative images of phospho-histone H3 (p-Hist H3) IHC on brain sections from the experiment described in C. Arrows point to and dotted line surrounds the location of metastatic cells in the brain. Scale bars, 100 μm. E, Quantitative analysis of the phospho-histone H3–positive cells within brain sections from the experiment described in C and represented in D. Immunostained sections were counted using the QuPath software, where positively stained cells are identified by setting a threshold for signal intensity (3+). Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 12 for Lenti-Con and n = 6 for S100A9i. F, Representative images of brain sections stained with an antibody against human S100A9. PC9-BrM cells were injected into the arterial circulation of immunodeficient mice via intracardiac injection. After metastatic signal was detected by bioluminescence imaging, treatment was started 25 days after tumor cell injection with either vehicle or osimertinib at 5 mg/kg body weight/day by oral gavage 5 days per week. Brain tissues were collected 2 months after tumor cell injection in the vehicle treatment group (Vehicle) 3 months after tumor cell injection in the osimertinib treatment group (minimal residual disease, or MRD) and 8 months after tumor cell injection in the osimertinib-treated relapse group (Relapse). Scale bars, 100 μm. Data are representative of 10 mice/group analyzed at each time point. G, Schematic representation of single-cell cloning from PC9-BrM cells. S100A9 high- and low-expressing single-cell progenies (SCP) are labeled as S100A9hi and S100A9lo, respectively. H, Immunoblot analysis of lysates from PC9-BrM–derived SCPs using antibodies against S100A8, S100A9, and β-actin (loading control). The data are representative of three independent experiments. I, Schematic representation of the brain metastasis assay to compare the ability of S100A9hi and S100A9lo SCPs to grow in the brain and generate metastases. J, Ex vivo photon flux of brains from mice injected with PC9-BrM–derived S100A9hi and S100A9lo SCPs was determined by bioluminescence imaging. Brains were collected from mice 7 weeks after tumor cell injection. Photon-flux scale is indicated below the images. K, Violin plots depicting normalized photon flux of brains imaged ex vivo from mice described in J. The normalized photon flux for brain tissue was calculated by dividing the photon flux from brain collected ex vivo by the total photon flux at day 0 (i.e., the day of injection) and multiplying that value by 100. Data are presented as mean values ± SEM. P values were determined by a two--tailed, unpaired Mann–Whitney test. N = 5 for S100A9hi; n = 4 for S100A9lo. L, Representative images of CK7 IHC on brain sections from mice injected with PC9-BrM–derived S100A9hi and S100A9lo SCPs. Brains were harvested from mice 7 weeks after tumor cell injection. Scale bars, 200 μm. M, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis (Met area) in the experiment described in L. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 5 for S100A9hi and n = 5 for S100A9lo.

S100A9-proficient cells promote postcolonization growth in the brain. A, Schematic representation of the experimental design to quantify seeding in the brain. PC9-Tr-BrM cells expressing either lenti-control (Lenti-Con) or S100A9i were injected into the arterial circulation of immunodeficient mice via intracardiac injection. Seven days later, brains were isolated, sectioned, and analyzed by IHC for human CK7. CK7-immunostained cancer cells were then counted to compare seeding of cancer cells in the brain parenchyma between experimental groups. B, Quantitative analysis of the experiment described in A. Tumor cells were counted in 10 sections of 20 μm each per brain. Data are presented as mean values ± SEM. The P value was determined by a two--tailed, unpaired Mann–Whitney test. N = 4 for Lenti-Con; n = 5 for S100A9i. ns, P value not significant. C, Schematic representation of the experimental design to analyze postcolonization growth in the brain. PC9-Tr-BrM cells expressing either Lenti-Con or S100A9i were injected into the arterial circulation of immunodeficient mice via intracardiac injection. At 7 weeks after injection, brain tissues were collected, and sections were analyzed by immunostaining for phospho-histone H3 (Ser10) to compare the number of mitotically active cancer cells between the experimental groups. D, Representative images of phospho-histone H3 (p-Hist H3) IHC on brain sections from the experiment described in C. Arrows point to and dotted line surrounds the location of metastatic cells in the brain. Scale bars, 100 μm. E, Quantitative analysis of the phospho-histone H3–positive cells within brain sections from the experiment described in C and represented in D. Immunostained sections were counted using the QuPath software, where positively stained cells are identified by setting a threshold for signal intensity (3+). Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 12 for Lenti-Con and n = 6 for S100A9i. F, Representative images of brain sections stained with an antibody against human S100A9. PC9-BrM cells were injected into the arterial circulation of immunodeficient mice via intracardiac injection. After metastatic signal was detected by bioluminescence imaging, treatment was started 25 days after tumor cell injection with either vehicle or osimertinib at 5 mg/kg body weight/day by oral gavage 5 days per week. Brain tissues were collected 2 months after tumor cell injection in the vehicle treatment group (Vehicle) 3 months after tumor cell injection in the osimertinib treatment group (minimal residual disease, or MRD) and 8 months after tumor cell injection in the osimertinib-treated relapse group (Relapse). Scale bars, 100 μm. Data are representative of 10 mice/group analyzed at each time point. G, Schematic representation of single-cell cloning from PC9-BrM cells. S100A9 high- and low-expressing single-cell progenies (SCP) are labeled as S100A9hi and S100A9lo, respectively. H, Immunoblot analysis of lysates from PC9-BrM–derived SCPs using antibodies against S100A8, S100A9, and β-actin (loading control). The data are representative of three independent experiments. I, Schematic representation of the brain metastasis assay to compare the ability of S100A9hi and S100A9lo SCPs to grow in the brain and generate metastases. J,Ex vivo photon flux of brains from mice injected with PC9-BrM–derived S100A9hi and S100A9lo SCPs was determined by bioluminescence imaging. Brains were collected from mice 7 weeks after tumor cell injection. Photon-flux scale is indicated below the images. K, Violin plots depicting normalized photon flux of brains imaged ex vivo from mice described in J. The normalized photon flux for brain tissue was calculated by dividing the photon flux from brain collected ex vivo by the total photon flux at day 0 (i.e., the day of injection) and multiplying that value by 100. Data are presented as mean values ± SEM. P values were determined by a two--tailed, unpaired Mann–Whitney test. N = 5 for S100A9hi; n = 4 for S100A9lo. L, Representative images of CK7 IHC on brain sections from mice injected with PC9-BrM–derived S100A9hi and S100A9lo SCPs. Brains were harvested from mice 7 weeks after tumor cell injection. Scale bars, 200 μm. M, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis (Met area) in the experiment described in L. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 5 for S100A9hi and n = 5 for S100A9lo.