Treatment with HIF-1α Antagonist PX-478 Inhibits Progression and Spread of Orthotopic Human Small Cell Lung Cancer and Lung Adenocarcinoma in Mice  Jörg J. Jacoby, PhD, Baruch Erez, DVM, Maria V. Korshunova, MS, Ryan R. Williams, MS, Kazuhisa Furutani, MD, PhD, Osamu Takahashi, MD, PhD, Lynn Kirkpatrick, PhD, Scott M. Lippman, MD, Garth Powis, DPhil, Michael S. O'Reilly, MD, Roy S. Herbst, MD, PhD  Journal of Thoracic Oncology  Volume 5, Issue 7, Pages 940-949 (July 2010) DOI: 10.1097/JTO.0b013e3181dc211f Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 HIF-1α is expressed in lung cancer cells. A, Western blot analysis of human NSCLC (A427, A549, H226, H322, H358, H441, H460, H1299, and PC14PE6) and small cell lung cancer (SCLC; H187 and N417) cell lines. Cells were grown in media, with or without 150 μM CoCl2, for 18 hours. Total cell lysates (50 μg) were evaluated by Western blot analysis for HIF-1α protein expression. B, NCI-H187 and PC14PE6 cells were treated with PX-478, with or without 150 μM CoCl2, for 16 hours. Total cell lysates (50 μg) were evaluated by Western blot analysis for HIF-1α protein expression. C, Immunohistochemical analyses of representative sections obtained from different primary lung tumors grown orthotopically in mice stained with an antibody directed against human HIF-1α. Journal of Thoracic Oncology 2010 5, 940-949DOI: (10.1097/JTO.0b013e3181dc211f) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 PX-478 treatment inhibits orthotopic growth (A, B) and increases survival (C) of human adenocarcinoma PC14PE6 and human small cell lung cancer (SCLC) NCI-H187 (D, E, F). A, PC14PE6 cells (1 × 106) were injected into the left lungs of nude mice; treatment with vehicle, PX-478 (10 or 20 mg/kg daily for 5 days), or cyclophosphamide (150 mg/kg 3 days a week for 1 week) was initiated 18 days after cell injection. Mice were killed when significant morbidity was observed, and the left lungs were weighed. B, A second experiment to evaluate different schedules of PX-478 and combination chemotherapy on tumor growth (left lung weight). Treatment was initiated 15 days after tumors had been injected with vehicle, paclitaxel (200 μg/mouse, weekly), or PX-478 (20 mg/kg, either daily for 5 days [QD × 5], 3 days a week [Monday, Wednesday, and Friday], or every third day [Q3D]), with or without paclitaxel, for the duration of the experiment. C, PC14PE6 (1 × 106) were injected into the left lungs of nude mice. Treatment with 20 mg/kg PX-478 or vehicle (daily for 5 days) was started 18 days after injection. Mice were monitored daily and killed when they became moribund. D, H187 cells (1 × 106) were injected into the left lungs of nude mice, and treatment with vehicle, PX-478 (10 or 20 mg/kg daily for 5 days), or cyclophosphamide (150 mg/kg 3 days a week for 1 week) was initiated 17 days after cell injection. Mice were killed when significant morbidity was observed, and the left lungs were weighed. E, A second experiment to evaluate different schedules of PX-478 and combination chemotherapy on tumor growth (left lung weight). Treatment was initiated 15 days after tumor injection with vehicle, paclitaxel (200 μg/mouse, weekly), or 20 mg/kg PX-478 daily for 5 days (QD × 5), 3 days a week (Monday, Wednesday, and Friday), or every third day (Q3D), with or without paclitaxel, for the duration of the experiment. F, H187 cells (1 × 106) were injected into the left lungs of nude mice. Treatment with 20 mg/kg PX-478 or vehicle (daily for 5 days) was started 17 days after injection. Mice were monitored daily and killed when they became moribund (Pac. = paclitaxel). Journal of Thoracic Oncology 2010 5, 940-949DOI: (10.1097/JTO.0b013e3181dc211f) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Treatment with PX-478 induces apoptosis but does not alter microvessel density or HIF-1α staining. A, Immunohistologic quantification of apoptosis, microvessel density, or HIF-1α staining in tissue derived from PC14PE6, NCI-N417, and NCI-H187 tumors after the last treatment with PX-478 (day 5 of treatment). Tissue sections were stained with an anticleaved caspase-3 antibody, anti-CD31, or anti-HIF-1α antibody. For apoptosis and microvessel density, the number of positive cells was determined by counting 12–16 high-power fields at 200× magnification. For HIF-1α, the mean percentages of positive tumor cells from three or four tumors are shown. B, Time-course experiment of HIF-1α expression. Mice were killed 0, 2, 4, 6, 8, or 16 hours after a single dose of 20 mg/kg PX-478 at day 21 after injection of 1 × 106 PC14PE6 cells into the left lung. The mean percentages of HIF-1α-positive tumor cells from three or four tumors and representative stained tissue sections are shown. Journal of Thoracic Oncology 2010 5, 940-949DOI: (10.1097/JTO.0b013e3181dc211f) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions