Fig 2: S100A9 is a key mediator of brain relapse in osimertinib-refractory lung cancer cells. A, Immunoblot analysis for inhibition of EGFR pathway activation in PC9-BrM and PC9-Tr-BrM cells treated with the indicated doses of osimertinib (Osi) and collected 6 hours after treatment. Antibodies against phospho-EGFR (Tyr1068), phospho-ERK (Thr202/Tyr204), total EGFR, total ERK, and ß-actin (loading control) were used. Data are representative of three independent experiments. B, Brain sections from mice injected with PC9-BrM cells described in Fig. 1A and B were immunostained using an antibody against phospho-EGFR. PC9-BrM cells were injected into the arterial circulation of immunodeficient mice via intracardiac injection to generate metastases. Treatment was administered starting 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 and continued until endpoint. The endpoint for vehicle-treated mice was 2 months after tumor cell injection (Vehicle). The endpoint for osimertinib-treated mice was 4 months after tumor cell injection for micrometastases (Micromet) and 8 months after tumor cell injection for relapsed metastatic lesion (Relapsed Met). For the osimertinib treatment group, mice were administered osimertinib by oral gavage. At 6 hours after treatment, mice were euthanized, and brain tissues were subsequently processed for histologic analysis. Representative images of IHC staining for phospho-EGFR in brain sections are shown. Arrows point to and dotted line surrounds the location of metastatic cells in the brain. Scale bars, 100 µm. C, The p-EGFR–immunostained brain sections described in B were quantitated using automated QuPath software to count p-EGFR–positive (pos.) cancer cells that were identified by setting a threshold for signal intensity (1+). Data are presented as mean values ± SEM. P values (indicated in the figures) were determined by a two-tailed, unpaired Mann–Whitney test: n = 10 for vehicle-treated mice, 14 for osimertinib-treated mice with micrometastases, and 10 for osimertinib-treated mice with relapsed metastases. Veh, vehicle. D, Schematic representation of the strategies used to compare PC9-BrM and PC9-Tr-BrM cells for differentially expressed proteins by quantitative label-free mass spectrometry and for differentially expressed genes by transcriptomics. E, Volcano plot shows the differentially expressed proteins between PC9-Tr-BrM and PC9-BrM cells identified by quantitative label-free mass spectrometry. Proteins with higher abundance in PC9-Tr-BrM cells compared with PC9-BrM cells have log2 fold changes with positive values and are labeled in red. N = 3 replicates per group. Data points referring to the top significantly differentially expressed proteins (S100A9 and S100A8) are labeled. F, Volcano plot of RNA-seq–based transcriptomic analysis shows the differentially expressed genes between PC9-Tr-BrM cells and PC9-BrM cells. Genes with significantly higher expression in PC9-Tr-BrM cells compared with PC9-BrM cells have log2 fold changes with positive values and are depicted in red. Genes with significantly lower expression in PC9-Tr-BrM cells compared with PC9-BrM cells have log2 fold changes with negative values and are depicted in gray. N = 3 replicates per group. Data points referring to the top significantly differentially expressed genes (S100A9 and S100A8) are labeled. G, Immunoblot analyses of lysates from BrM and Tr-BrM cells from both PC9- and H1650-derived models using antibodies against S100A9 and ß-actin (loading control). The data are representative of three independent experiments. H, S100A9 expression was determined by qRT--PCR analysis of PC9- and H1650-derived BrM and Tr-BrM cells. GAPDH was measured as an internal control. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test: n = 6 for PC9-BrM, n = 6 for PC9-Tr-BrM, n = 4 for H1650-BrM, and n = 5 for H1650-Tr-BrM. Rel., relative. I, Immunoblot analyses of lysates from PC9- and H1650-derived Tr-BrM cells infected with viruses expressing either a control gRNA (Lenti-Con) or an S100A9-specific gRNA (referred to as “S100A9i” throughout the figures). The indicated antibodies were used to confirm the loss of S100A9 protein expression following CRISPR/dCas9-mediated gene repression. ß-Actin served as a protein loading control. Data are representative of three independent experiments. J,Ex vivo photon flux of brains from mice injected with PC9- or H1650-derived Tr-BrM cells expressing either Lenti-Con or S100A9i was determined by bioluminescence imaging. Mice were collected at 7 weeks after tumor cell injection. The photon-flux scale is indicated on the right side. K, Violin plots depicting normalized photon flux of brains imaged ex vivo from the 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. For PC9 Tr-BrM, n = 10 for Lenti-Con and n = 9 for S100A9i. For H1650-Tr-BrM, n = 6 for Lenti-Con and n = 7 for S100A9i. L, Representative images of CK7 IHC on brain sections from mice injected with either PC9-derived Tr-BrM Lenti-Con–expressing (left) or S100A9i-expressing (right) cells in the top row, or H1650-derived Tr-BrM Lenti-Con–expressing (left) or S100A9i-expressing (right) cells in the bottom row. Brains were harvested from mice 7 weeks after tumor cell injection. Scale bars, 500 µm. M, Quantitative analysis of the percentage of brain sections covered by metastasis from the experiment described in L. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM, n = 4 for Lenti-Con and n = 4 for S100A9i. For H1650-Tr-BrM, n = 6 for Lenti-Con and n = 3 for S100A9i.

Fig 3: High S100A9 expression is associated with brain metastasis and shorter PFS in patients with osimertinib-treated lung cancer. A, Schematic representation of the analysis of patient samples for S100A9 expression in cancer cells. S100A9 immunostaining was performed on tissue specimens (biopsies/resected material) from 29 patients with lung cancer with a validated EGFR mutation that were obtained prior to osimertinib treatment. The immunostained samples were scored by independent pathologists as either S100A9-positive (any percentage of clear, positive intracellular S100A9 staining in cancer cells) or S100A9-negative (no detectable S100A9 staining in cancer cells). neg, S100A9-negative; pos, S100A9-positive. B, Graphical representation of the association between S100A9 expression in the patient tissue specimens described in A and the development of brain metastasis (met) for 26 patients with a clinical annotation for the presence or absence of brain metastasis at diagnosis (three of 29 patients had unknown brain metastasis status at diagnosis). The P value was determined by a ?2 test: n = 10 samples from patients with brain metastasis and n = 16 samples from patients without brain metastasis. C, Distribution of patients on first-, second-, and third-line osimertinib (Osi) treatment from the 29-patient cohort described in A. D, Kaplan–Meier plot for the PFS of osimertinib-treated patients from the combined cohort described in A. Data were analyzed using the log-rank test: ?2 = 10.74, degrees of freedom (d.f.) = 1, P = 0.0001, n = 29 patients. Patients who had not progressed at the time of analysis were censored. E, Kaplan–Meier plot for PFS of the osimertinib-treated patients described in A and C. Data were analyzed using the log-rank test. For first-line osimertinib-treated patients: ?2 = 6.011, d.f. = 1, P = 0.0106, n = 17; for second- and third-line osimertinib-treated patients: ?2 = 4.015, d.f. = 1, P = 0.0451, n = 12. Patients who had not progressed at the time of analysis were censored.

Fig 4: 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 ß-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.

Fig 5: Confirmation of S100A9, p62, COL17A1, and LC3 expression in olaparib resistant cells. (A) Western blot analysis of S100A9, p62, COL17A1, and p62 associated protein LC3-I, II in both parental and resistant SUM159 and MDA468 cells. (B) Effect of autophagy stimulation on p62 and LC3 expression. Parental and resistant SUM159 cells were starved for indicated periods (upper panel) or treated with two different doses (5 µM and 10 µM) of rapamycin for 24 hrs (lower panel). The levels of p62 or LC3 were measured by western blotting. Rap, rapamycin and starv, starvation. (C) Effect of olaparib withdrawal on LC3 and p62 levels. SUM159-R cells were maintained in olaparib-free media for 3 days and 10 days or in olaparib-containing (25 µM) medium (ola).). P, parental; R, resistant; Starv., starvation; Rap, rapamycin; and Ola, olaparib.