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. Author manuscript; available in PMC: 2012 Oct 11.
Published in final edited form as: Oncogene. 2011 Jul 4;31(6):706–715. doi: 10.1038/onc.2011.275

Figure 4. The PI3-kinase inhibitor PIK-75 blocks HRGβ1 induced chemotaxis, in vivo invasion and total tumor cell motility.

Figure 4

A. In vitro chemotaxis is inhibited by PIK-75. Migration of ErbB3WT cells in response to a gradient of either 1.25 nM HRGβ1 (black bar) or 1.25 nM HRGβ1 + 0.2 μM PIK-75 (patterned bar). *: p< 0.0001. Data are mean and SEM of measurements from 3 independent experiments.

B. In vivo invasion is inhibited by PIK-75. Microneedles containing 50 nM HRGβ1or 50 nM HRGβ1 + 1 μM PIK-75 were inserted into the primary tumor and invasive cells were collected and counted. Data are mean and SEM from 6 needles for HRGβ1 and 10 needles for HRGβ1 + PIK-75 from 3 tumors for each condition. *: p< 1 × 10-5.

C. In vivo invasion stimulated by 50 nM HRGβ1 is inhibited by systemic application of PIK-75. Mice bearing ErbB3WT tumors were injected i.p. either with vehicle control (black bar) or with 1.5 mg PIK-75 (pattern) 3 hours prior to assay. Microneedles containing 50 nM HRGβ1 were inserted into primary tumors and invasive cells were quantified as described in the methods.*: p< 0.00035. Data are means and SEM of 4-8 measurements from 4 tumors per condition.

D. Cell motility in vivo using intravital imaging. Mice bearing tumors were injected i.p. either with vehicle control (black bar) or with 1.5 mg PIK-75 (pattern) 3 hours prior to imaging. Tumors were exposed by skin-flap surgery and time-lapse z-series were acquired. The average total cell motility was determined per 40 μm z-stack (5 sections imaged at 10 μm intervals).

Top: Data are means and SEM of 8 (vehicle control) and 15 (PIK-75) separate z stacks from 3 mice per condition. *: p< 0.016

Bottom: Representative panels from tumors of animals injected with (i) MTLn3-ErbB3--GFP vehicle control or (ii) PIK-75 3 hours prior to imaging. Cells are green with collagen fibers detected by second harmonic scattering in purple. Frames are 6 minutes apart, arrow shows migrating tumor cell; * indicates a single cell. Scale bar = 25 μm.

E. Length/width ratio comparison. Top: representative images Bottom: Mean and SEM of 40-50 cells from 3 tumors per condition, 5 slices analyzed per field. Scale bar = 25 μm.*: p<0.002