Supplementary MaterialsS1 Fig: Fibroblasts stimulate the invasive properties of PANC-1 cells. per condition, d). Stars indicate the ECM degradation caused by PANC-1 cells. Scale bar = 10m. Graphs represent averages SEM from 3 independent experiments. **p 0.01. (e-j) CM from fibroblasts promotes ECM degradation by PANC-1 cells. PANC-1 cells were seeded onto green fluorescent gelatin-coated coverslips in DMEM only (e) or CM collected from HFs (f), hPSCs (g), or RFs (h), and matrix degradation was quantified after 8 h. In the presence of CM from stroma, PANC-1 cells start degrading the gelatin substrate, quantified both with the percentage of cells degrading matrix (100 cells per condition (i) and the area degraded per cell in PANC-1 cells (10 cells per condition (j), compared to the DMEM control. (k) Invadopodia were scored by quantifying actin puncta that colocalized with regions of matrix degradation. Scale bar = 10m. Graphs represent averages SEM from 3 independent experiments. *p 0.05. **p 0.01.(TIF) pone.0248111.s001.tif (970K) GUID:?8EBBE39E-D47E-4B61-BD96-5AB61E80BB6D S2 Fig: Conditioned media from RFs stimulates the ECM remodeling ability of pancreatic cancer cells. (a-h) Representative fluorescence images showing that CM from RFs dramatically enhanced gelatin degradation by DanG (a-b), HPAF-II (c-d), CFPAC (e-f), and MBA-MD-231 (g-h) cells over an 8 h Col4a3 period compared to the corresponding DMEM controls. Scale bar = 10m. (i-j) Bar graphs showing quantification of gelatin degradation Ulipristal acetate by the various PDAC tumor cells listed above. Quantification shows a marked increase both in the percent of PDAC cells degrading the gelatin matrix (100 cells per condition, i) and the degradation area per cell area (10 cells per condition, j). Graph represents Ulipristal acetate averages SEM from 3 independent experiments. *p 0.05. **p 0.01. n.s, not statistically significant.(TIF) pone.0248111.s002.tif (6.3M) GUID:?D54E68B5-5C19-4419-8616-775A1E9CC326 S3 Fig: The ECM remodeling capacity of pancreatic cancer cells correlates with the level of MMP2 secreted into the culture media by stromal cells. (a-h) Fluorescence images of BxPC3 tumor cells seeded on green fluorescent gelatin-coated coverslips and cultured with (a) DMEM containing 10% FBS for 8 h before fixation, (b) the MMP inhibitor BB-94 (2 M) for 8 h, (c-f) CM from different stromal cells for 8 h after BB-94 washout. (g,h) Graphs depicting matrix degradation by cells described above and quantified as either the percentage of BxPC3 cells degrading matrix (100 cells per condition, g) or the degradation area per cell area (10 cells per condition, h). These data suggest a direct correlation between the level of MMP2 in the stromal cell CM and the matrix degradation induced by the addition of this CM to the BxPC3 cells. Scale bar = 10m. Bar graph Ulipristal acetate represents averages SEM from 3 independent experiments. **p 0.01. n.s, not statistically significant. (i-q) MMP2 levels in CM collected from cancer cells correlates with the capacity to promote matrix degradation in recipient BxPC3 cells. (i) Zymography demonstrating MMP2 activity in 5 different PDAC cell lines compared to HFs. (j-o) Representative images of BxPC3 degrading matrix in response to CM derived from other tumor cells represented in the above zymogram. Note that the tumor cells secreting the lower amounts of MMP2 into the CM induce the least degradation by the recipient BxPC3 cells. Scale bar = 10m. (p,q) Bar graph quantifying BxPC3 matrix degradation in response to CM from the distinct tumor cells. The percentage of BxPC3 cells degrading matrix was determined with 100 cells per condition and the degradation area per cell area was quantified with 10 cells per condition. Graphs represent averages SEM from 3 independent experiments.(TIF) pone.0248111.s003.tif (5.3M) GUID:?B349706B-4640-4E80-B88E-599F401D79B8 S4 Fig: The depletion of MMP2 from stromal cell conditioned medium reduces the ECM remodeling capacity of BxPC3 cells. (a) Western blot of HF cells treated with control siRNAs or siRNAs to reduce MMP2 levels. (b) Zymogram showing loss of MMP2 in the siRNA-treated cells described in (a). (c-d) Fluorescence images of BxPC3 cells plated on green fluorescent gelatin-coated coverslips and incubated 8 h with CM collected from HFs cells treated with control siRNAs or siRNAs to reduce MMP2 levels. Phalloidin staining of actin was used to show cell borders (c, d). Scale bar = 10m. (e,f) Quantitation of the experiment described above. Depletion of MMP2 from the HF-CM.