Cross-talk between human airway epithelial cells and 3T3-J2 feeder cells involves partial activation of human MET by murine HGF

<div><p>There is considerable interest in the <i>ex vivo</i> propagation of primary human basal epithelial stem/progenitor cells with a view to their use in drug development, toxicity testing and regenerative medicine. These cells can be expanded in co-culture with mitotically inactivated 3T3-J2 murine embryonic feeder cells but, similar to other epithelial cell culture systems employing 3T3-J2 cells, the aspects of cross-talk between 3T3-J2 cells and human airway basal cells that are critical for their expansion remain largely unknown. In this study, we investigated secreted growth factors that are produced by 3T3-J2 cells and act upon primary human airway basal cells. We found robust production of hepatocyte growth factor (HGF) from fibroblast feeder cells following mitotic inactivation. Consistent with the limited cross-species reactivity of murine HGF on the human HGF receptor (MET; HGFR), MET inhibition did not affect proliferative responses in human airway basal cells and HGF could not replace feeder cells in this culture system. However, we found that murine HGF is not completely inactive on human airway epithelial cells or cancer cell lines but stimulates the phosphorylation of GRB2-associated-binding protein 2 (GAB2) and signal transducer and activator of transcription 6 (STAT6). Although HGF induces phosphorylation of STAT6 tyrosine 641 (Y641), there is no subsequent STAT6 nuclear translocation or STAT6-driven transcriptional response. Overall, these findings highlight the relevance of cross-species protein interactions between murine feeder cells and human epithelial cells in 3T3-J2 co-culture and demonstrate that STAT6 phosphorylation occurs in response to MET activation in epithelial cells. However, STAT6 nuclear translocation does not occur in response to HGF, precluding the transcriptional activity of STAT6.</p></div

Activation of the HGF receptor (MET) in primary human airway basal epithelial cells by 3T3-J2 murine embryonic fibroblast feeder cell-conditioned medium.

<p>(A) Receptor tyrosine kinase array analysis of primary human airway basal cells stimulated for 30 minutes with 3T3-J2 feeder cell-conditioned medium. Specific phosphorylation of the hepatocyte growth factor (HGF) receptor (HGFR/MET) at Y1234/Y1235 was observed in cells stimulated with 3T3-J2-conditioned medium both in the presence and absence of Y-27632, a RHO-associated protein kinase (ROCK) inhibitor. The three duplicate spots in the top left, top right and bottom left of each array are assay reference spots. This array was performed using one donor cell culture. (B) Schematic representation of MET receptor structure showing relevant phosphorylation sites. Sema = semaphorin; PSI = plexins-semaphorins-integrins; IPT = immunoglobulins-plexins-transcription factor. (C) Western blot confirmation in independent lysates of MET phosphorylation following stimulation of primary human airway basal cells with 3T3-J2 feeder cell-conditioned medium for 30 minutes. Y indicates the presence of 5 μM Y-27632 in the culture medium. The blots shown are representative of experiments performed on three donor cell cultures.</p

mHGF is secreted by 3T3-J2 fibroblasts following mitotic inactivation but primary human airway basal cell MET does not mediate important aspects of co-culture expansion.

<p>(A) ELISA quantification of hepatocyte growth factor (HGF) secreted into culture medium by 3T3-J2 feeder cells following mitotic inactivation with mitomycin C. Medium was collected and replaced with fresh medium after 24 and 48 hours (n = 4; mean +/- SEM; ** indicates p<0.01 using a one-way ANOVA with Holm-Sidak’s correction for multiple comparisons). (B) Flow cytometric analysis of EdU uptake in primary human airway basal cells treated with either epithelial culture medium alone (+Y), 3T3-J2-conditioned epithelial growth medium (CM+Y) or the same medium containing 100 nM PF-0421903 (CM+Y+PF), a small molecule MET inhibitor, for 12 hours (CM+Y+PF; n = 4 donor cultures performed in triplicate repeats; mean +/- SEM; * indicates p<0.05 using a one-way ANOVA with Holm-Sidak’s correction for multiple comparisons). (C) Colony-forming efficiency of primary human airway basal cells grown on 3T3-J2 feeder cells in the presence of 5 μM Y-27632 and either 100 nM PF-0421903 or a vehicle control for 10 days (n = 5 donor cultures; ns indicates not significant using a paired t test; representative wells from one donor are shown in images). (D) Colony-forming efficiency of primary human airway basal cells grown on 3T3-J2 feeder cells without EGF or Y-27632 for 10 days. 3 ng/ml or 50 ng/ml mHGF or hHGF was added as indicated (n = 3 donor cultures; * represents p<0.05 and ** represents p<0.01 using a using a two-way ANOVA with Holm-Sidak’s correction for multiple comparisons to compare each concentration of HGF to the vehicle control group; representative wells from one donor are shown in images). (E) MTT assay using primary human airway epithelial cells cultured in medium without EGF or Y-27632 for 10 days. 3 or 50 ng/ml mHGF or hHGF was added as indicated (n = 3 donor cultures; ** represents p<0.01 and *** represents p<0.001 using a using a two-way ANOVA with Holm-Sidak’s correction for multiple comparisons to compare each concentration of HGF to the vehicle control group). (F) Cell counts comparing primary human airway basal cell number in 3T3-J2 co-culture in medium containing Y-27632 and vehicle protein (3T3+Y+Vehicle) with medium alone (i.e. no feeder cells; Y+Vehicle) and medium plus either 3 ng/ml or 50 ng/ml hHGF. Cells were analysed after 24, 48 or 72 hours. In an additional condition, after 72 hours cells were passaged to the same density as at time 0 and analysed after a further 96 hours (n = 3 donor cultures performed in triplicate repeats; mean +/- SEM; **** indicates p<0.0001 using a two-way ANOVA with Holm-Sidak’s correction for multiple comparisons to compare media compositions with the 3T3+Y group).</p

3T3-J2-conditioned medium and hHGF stimulate MET, GAB2 and STAT6 phosphorylation in primary human airway basal cells.

<p>(A) Western blot analysis of MET, GRB2-associated-binding protein 2 (GAB2) and signal transducer and activator of transcription 6 (STAT6) phosphorylation status following stimulation of primary human airway basal cells with basal or 3T3-J2-conditioned medium (CM) for 30 minutes. Y indicates the presence of 5 μM Y-27632 and PF indicates 100 nM PF-04217903. Blots are from one donor cell culture and are representative of experiments performed in three further donor cultures. (B) Western blot analysis of MET, GAB2 and STAT6 phosphorylation status in primary human airway basal cells stimulated with either 3 ng/ml or 50 ng/ml recombinant mouse (mHGF) or recombinant human hepatocyte growth factor (hHGF) for 30 minutes. Blots are from one donor cell culture and are representative of experiments performed in two further donor cultures. (C) Dose-response western blot analysis of HGF-induced STAT6 phosphorylation in primary human airway basal cells stimulated with the indicated amount of hHGF for 30 minutes. This result was replicated in a second donor cell culture.</p

An MR image of a low tumor cell seeding model mouse lung the corresponding whole lung composite H & E stained section, a blown up area and sizing of tumor (white box), and CD34 immunohistochemistry image.

<p>A) a single enhancing tumour (at 25 minutes) with corresponding CD34 image (C), B) two visible tumors (pre contrast image) with corresponding CD34 images left (D) and right (E).</p

MR images of a high tumor cell seeding model mouse at day 14 over the 35 minutes for high dose Gd.DTPA (white arrowsheads denote visible tumors) (A), and the corresponding mean values for the percentage increase in observable tumors compared to that of high tumor cell seeding mice at day 14 for eqMRI (B).

<p>Values are mean ± s.e.m. * p = <0.05 from high dose.</p

Low tumor cell seeding model mean values for signal-to noise (SNR) and the percentage increase in observed tumours for eqMRI.

<p>A) SNR of tumors already visible on pre contrast images, B) SNR of tumors that were visible due to Gd enhancement from 15 minutes onwards, C) the percentage increase in observed tumors by Gd enhancement at each time point compared to pre contrast. Values are mean ± s.e.m. * p = <0.05 from pre (A and B) or from 15 min (C).</p

Experimental plan showing timings of bolus and infusion as well as scan times.

<p>The bars above are an assumed grey scale representation of gadolinium concentration within the blood where black is high and white low concentration.</p