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

Figure 6.

Figure 6. Osimertinib-refractory cancer cells are sensitive to pan-RAR inhibition. A, Schematic representation of the treatment of PC9- and H1650-derived Tr-BrM cells with vehicle, osimertinib alone, a pan-RAR antagonist (AGN194310) alone, or AGN194310 in combination with osimertinib. At 25 days after tumor cell injection, after confirmation of metastatic signal, mice were administered long-term treatment with either (i) vehicle (Veh), (ii) AGN194310 (Pan-RARi; 0.5 mg/kg body weight/day), (iii) osimertinib (Osi; 5 mg/kg body weight/day), or (iv) AGN194310 (0.5 mg/kg body weight/day) plus osimertinib (5 mg/kg body weight/day) by oral gavage 5 days per week until the endpoint (7 weeks after tumor cell injection). B, Ex vivo photon flux of posttreatment brains from the experiment described in A was determined at endpoint by bioluminescence imaging. The photon-flux scale is indicated on the right. C, Violin plots depicting normalized photon flux of brains imaged ex vivo from the mice described in B. 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 mice: n = 8 for vehicle; n = 8 for osimertinib; n = 7 for pan-RARi; and n = 7 for osimertinib plus pan-RARi. For H1650-Tr-BrM mice: n = 12 for vehicle; n = 8 for osimertinib; n = 10 for pan-RARi; and n = 6 for osimertinib plus pan-RARi. ns, P value not significant. D, Representative images of CK7 IHC on posttreatment brain sections collected at endpoint from the experiment described in A. Scale bars, 200 μm for top (PC9-Tr-BrM) and 100 μm for bottom (H1650-Tr-BrM). E, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis (Met) shown in D. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM mice: n = 11 for vehicle; n = 9 for osimertinib; n = 4 for pan-RARi; and n = 5 for osimertinib plus pan-RARi. For H1650-Tr-BrM mice: n = 6 for vehicle; n = 6 for osimertinib; n = 9 for pan-RARi; and n = 6 for osimertinib plus pan-RARi. F, Schematic representation of the experiment testing the effect of RAR gene knockdown on brain metastasis development. Mice were injected with either PC9- or H1650-derived Tr-BrM cells with one of two sets of shRNA-mediated stable dual knockdown of RARα and RARγ (sh-RARα + γ), or control shRNA (sh-Con), via intracardiac injection. Experiments involving shRNA set #1 are shown in G–J, whereas experiments involving shRNA set #2 are shown in Supplementary Fig. S6L–S6O. Mice were euthanized 7 weeks after tumor cell injection, and brains were collected for analysis. G, Ex vivo photon flux of posttreatment brains from the experiment described in F was determined at endpoint by bioluminescence imaging. The photon-flux scale is indicated on the right. H, Violin plots depicting normalized photon flux of brains imaged ex vivo from the mice represented in G. 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 = 7 for sh-Con; n = 5 for sh-RAR. For H1650-Tr-BrM: n = 8 for sh-Con; n = 6 for sh-RAR. I, Representative images of CK7 IHC on posttreatment brain sections collected at endpoint from the experiment described in F. Scale bars, 100 μm for all images. J, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis shown in I. 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 sh-Con; n = 10 for sh-RAR. For H1650-Tr-BrM: n = 5 for sh-Con; n = 5 for sh-RAR.

Osimertinib-refractory cancer cells are sensitive to pan-RAR inhibition. A, Schematic representation of the treatment of PC9- and H1650-derived Tr-BrM cells with vehicle, osimertinib alone, a pan-RAR antagonist (AGN194310) alone, or AGN194310 in combination with osimertinib. At 25 days after tumor cell injection, after confirmation of metastatic signal, mice were administered long-term treatment with either (i) vehicle (Veh), (ii) AGN194310 (Pan-RARi; 0.5 mg/kg body weight/day), (iii) osimertinib (Osi; 5 mg/kg body weight/day), or (iv) AGN194310 (0.5 mg/kg body weight/day) plus osimertinib (5 mg/kg body weight/day) by oral gavage 5 days per week until the endpoint (7 weeks after tumor cell injection). B,Ex vivo photon flux of posttreatment brains from the experiment described in A was determined at endpoint by bioluminescence imaging. The photon-flux scale is indicated on the right. C, Violin plots depicting normalized photon flux of brains imaged ex vivo from the mice described in B. 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 mice: n = 8 for vehicle; n = 8 for osimertinib; n = 7 for pan-RARi; and n = 7 for osimertinib plus pan-RARi. For H1650-Tr-BrM mice: n = 12 for vehicle; n = 8 for osimertinib; n = 10 for pan-RARi; and n = 6 for osimertinib plus pan-RARi. ns, P value not significant. D, Representative images of CK7 IHC on posttreatment brain sections collected at endpoint from the experiment described in A. Scale bars, 200 μm for top (PC9-Tr-BrM) and 100 μm for bottom (H1650-Tr-BrM). E, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis (Met) shown in D. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM mice: n = 11 for vehicle; n = 9 for osimertinib; n = 4 for pan-RARi; and n = 5 for osimertinib plus pan-RARi. For H1650-Tr-BrM mice: n = 6 for vehicle; n = 6 for osimertinib; n = 9 for pan-RARi; and n = 6 for osimertinib plus pan-RARi. F, Schematic representation of the experiment testing the effect of RAR gene knockdown on brain metastasis development. Mice were injected with either PC9- or H1650-derived Tr-BrM cells with one of two sets of shRNA-mediated stable dual knockdown of RARα and RARγ (sh-RARα + γ), or control shRNA (sh-Con), via intracardiac injection. Experiments involving shRNA set #1 are shown in G–J, whereas experiments involving shRNA set #2 are shown in Supplementary Fig. S6L–S6O. Mice were euthanized 7 weeks after tumor cell injection, and brains were collected for analysis. G,Ex vivo photon flux of posttreatment brains from the experiment described in F was determined at endpoint by bioluminescence imaging. The photon-flux scale is indicated on the right. H, Violin plots depicting normalized photon flux of brains imaged ex vivo from the mice represented in G. 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 = 7 for sh-Con; n = 5 for sh-RAR. For H1650-Tr-BrM: n = 8 for sh-Con; n = 6 for sh-RAR. I, Representative images of CK7 IHC on posttreatment brain sections collected at endpoint from the experiment described in F. Scale bars, 100 μm for all images. J, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis shown in I. 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 sh-Con; n = 10 for sh-RAR. For H1650-Tr-BrM: n = 5 for sh-Con; n = 5 for sh-RAR.