Journal: Scientific Reports

Article Title: Discoidin Domain Receptors, DDR1b and DDR2, Promote Tumour Growth within Collagen but DDR1b Suppresses Experimental Lung Metastasis in HT1080 Xenografts

doi: 10.1038/s41598-020-59028-w

Figure Lengend Snippet: Doxycycline regulated expression of recombinant DDR1b and DDR2 in HT1080 cells. HT-DDR1b ( A ) and HT-DDR2 ( B ) cells processed to express or repress recombinant DDRs, as described in the Methods section, were incubated in serum-free media supplemented with or without 20 μg/ml of COL1 for various times at 37 °C. At the end of the incubation period, the cells were lysed in RIPA buffer and equal protein amounts per lane (30 µg) were resolved by reducing SDS-PAGE followed by immunoblot analyses using antibodies to phosphorylated DDR1b (Y513) ( A , middle panel) or DDR2 (Y740) ( B , middle panel). The same blots were then stripped and reprobed with antibodies against total DDRs (upper panels), and against β-actin, as loading control (lower panels). Control (Ctrl.): cells (harvested at 48 h), which were incubated with or without DOX but not stimulated with COL1. CTF, C-terminal fragment of DDR1b; pCTF, pDDR1b, and pDDR2 refers to the phosphorylated protein. Full-length blots are presented in Supplementary Fig. .

Article Snippet: Rat-tail COL1 (regular and high concentration) was purchased from Discovery Labware Inc., CorningTM (Bedford, MA; Cat # 354236, regular; and # 354249, high concentration).

Techniques: Expressing, Recombinant, Incubation, SDS Page, Western Blot, Control

Journal: Scientific Reports

Article Title: Discoidin Domain Receptors, DDR1b and DDR2, Promote Tumour Growth within Collagen but DDR1b Suppresses Experimental Lung Metastasis in HT1080 Xenografts

doi: 10.1038/s41598-020-59028-w

Figure Lengend Snippet: DDRs and COL1 promote the growth rate of HT1080 cells in vivo but not in vitro . (A,B) Volumes of ±DDR/+COL1 tumours as a function of time. HT-DDR1b ( A ) and HT-DDR2 ( B ) cells were incubated for two days with or without DOX to repress or induce DDR expression, respectively. Then, the cells were harvested and mixed with an ice-cold solution of COL1 (2 mg/ml, final concentration), as described in the Methods section. Mice fed a regular or a DOX-supplemented diet were inoculated s.c. with 1 × 10 6 cells/mouse in 100 μl of the COL1/cell suspension. Number of mice for each group is provided in Supplementary Table . Tumours were measured every 2–3 days, and tumour volumes were calculated. Tumour growth rates were determined, as described in the Methods section. ( C , D ) Expression and activation of DDRs in ±DDR/+COL1 tumour extracts. Tumour extracts derived from representative mice harbouring either HT-DDR1b ( C ) and HT-DDR2 ( D ) ±DOX tumours (n = 4 in each group) were resolved by reducing SDS-PAGE followed by immunoblot analyses using antibodies against phosphorylated DDR1b (Y513) ( C , middle panel) or DDR2 (Y740) ( D , middle panel). The blots were then stripped and reprobed with antibodies against total DDRs (upper panels), and then reprobed for β-actin (lower panels). pCTF, phosphorylated C-terminal fragment of DDR1b. Asterisk indicates a non-specific band. Full-length blots are presented in Supplementary Fig. . ( E , F ) In vitro cell proliferation of ±DDR -expressing HT1080 cells in 2D COL1. HT-DDR1b ( E ) and HT-DDR2 ( F ) cells were incubated with or without DOX for three days, and then 2 × 10 4 cells/well were seeded on 24-well plates coated with fibrillar COL1 (100 μg/well), in triplicates, in complete media. At various time points, the cells were detached and counted with a Coulter counter. Results represent the average of three independent experiments.

Article Snippet: Rat-tail COL1 (regular and high concentration) was purchased from Discovery Labware Inc., CorningTM (Bedford, MA; Cat # 354236, regular; and # 354249, high concentration).

Techniques: In Vivo, In Vitro, Incubation, Expressing, Concentration Assay, Suspension, Activation Assay, Derivative Assay, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Discoidin Domain Receptors, DDR1b and DDR2, Promote Tumour Growth within Collagen but DDR1b Suppresses Experimental Lung Metastasis in HT1080 Xenografts

doi: 10.1038/s41598-020-59028-w

Figure Lengend Snippet: DDRs promote HT1080 tumour growth rate only when cells are co-inoculated with COL1. ( A , B ) Volumes of +DDR/±COL1 tumours as a function of time. HT-DDR1b ( A ) and HT-DDR2 ( B ) cells were incubated for two days without DOX to induce DDR1b or DDR2 expression. Then, the cells were harvested, and a fraction of the cells was mixed with an ice-cold solution of rat-tail COL1 (2 mg/ml, final concentration) whereas the other fraction was mixed with serum-free media. Mice fed a regular diet, were subcutaneously inoculated with 100 μl of the cell suspension (with or without COL1) containing 1 × 10 6 cells. Number of mice for each group is provided in Supplementary Table . Tumours were measured every 2–3 days, and tumour volumes were calculated. Tumour growth rates were determined, as described in the Methods section. Note: The +DDR/−COL1 groups here are the+DDR groups plotted in Fig. , and the +DDR/+COL1 groups are also plotted in Fig. . ( C , D ) Expression and activation of DDRs in +DDR/±COL1 tumour extracts. Tumour extracts of +DDR1b/±COL1 ( C ) and +DDR2/±COL1 ( D ) tumours (n = 5 for each group) in RIPA buffer were resolved by reducing SDS-PAGE followed by immunoblot analyses using antibodies against phosphorylated DDR1b (Y513) ( C , middle panel) or DDR2 (Y740) ( D , middle panel). The blots were then stripped and reprobed with antibodies to total DDRs (upper panels), and then reprobed for β-actin as loading control (lower panels). Blots were quantified as described in the Methods section. Scatter plots show median with interquartile range where each dot represents an individual tumour. Mann-Whitney U test was performed for statistical analyses. Full-length blots are presented in Supplementary Fig. . ( E , F ) In vitro cell proliferation of +DDR-expressing HT1080 cells in 2D COL1 vs. plastic. HT-DDR1b ( E ) and HT-DDR2 ( F ) cells were incubated without DOX for 3 days and then 2 × 10 4 cells/well were seeded on 24-well plates coated with or without fibrillar COL1 (100 μg/well), in triplicates, in complete media. At various time points, the cells were detached and counted with a Coulter counter. Results represent the average of three independent experiments and data plotted as described in Methods. Note: The +DDR/−COL1 groups here are the +DDR groups plotted in Fig. , and the +DDR/+COL1 groups are also plotted in Fig. . ( G , H ) Expression and activation of DDRs in HT1080 cells ±DDRs cultured for 5 days in 2D COL1 or plastic. HT-DDR1b ( G ) and HT-DDR2 ( H ) cells were incubated for three days with (+) or without (−) DOX to repress or induce DDR1b or DDR2 expression, respectively. Then, 2 × 10 4 cells/well were seeded on 24-well plates, on uncoated wells (−) or wells coated with fibrillar COL1 (100 μg/well) (+), in triplicates. The cells were then cultured in complete media for 5 days, and then lysed in RIPA buffer. Equal protein amounts per lane (20 µg) were resolved by reducing 7.5% SDS-PAGE followed by immunoblot analyses using antibodies to phosphorylated DDR1b (Y513) (pDDR1) or DDR2 (Y740) (pDDR2). The same blots were then stripped and reprobed with antibodies against total DDRs (middle panels), and against β-actin, as loading control (lower panels). Asterisks indicate non-specific bands. Full-length blots are presented in Supplementary Fig. .

Article Snippet: Rat-tail COL1 (regular and high concentration) was purchased from Discovery Labware Inc., CorningTM (Bedford, MA; Cat # 354236, regular; and # 354249, high concentration).

Techniques: Incubation, Expressing, Concentration Assay, Suspension, Activation Assay, SDS Page, Western Blot, Control, MANN-WHITNEY, In Vitro, Cell Culture

Journal: Scientific Reports

Article Title: Discoidin Domain Receptors, DDR1b and DDR2, Promote Tumour Growth within Collagen but DDR1b Suppresses Experimental Lung Metastasis in HT1080 Xenografts

doi: 10.1038/s41598-020-59028-w

Figure Lengend Snippet: Impact of DDRs on Hippo pathway components in HT1080 ± DDR/+COL1 tumours. ( A–H ) Immunoblots of Hippo core components. Extracts of HT-DDR1b tumours ( A , G ) −DDR1b and +DDR1b, n = 4 and n = 6 each, respectively; ( C , E ) ±DDR1b, n = 4 each) and HT-DDR2 tumours ( B , F ) ±DDR2, n = 4 each; ( D , H ) ±DDR2, n = 5 each) that were co-inoculated with COL1, and harvested at time of sacrifice, were resolved by reducing SDS-PAGE followed by immunoblot analyses using antibodies to the indicated Hippo pathway components: YAP-1, pYAP1-S127, and pYAP1-S397 ( A , B ); MST1 and MST2 ( C , D ); LATS1 ( E , F ); and KIBRA ( G , H ). Blots were probed with the indicated antibodies and quantified as described in the Methods section. Note: in panels A and B , the blots for pYAP1-S127 and pYAP1-S397 were each run in separate blots with the same tumour extract samples. Each blot was then stripped and reprobed for total YAP1. Then, the relative levels of phosphorylated to total YAP1 levels were each quantified with their respective blot. However, in the panels only one total YAP1 blot for DDR1b or DDR2 is shown. In panel B , the +DDR2 blot for pYAP1-S127 comprises samples that were run in the same blot. However, an empty lane in the middle was cropped and it is now indicated by a vertical black line. Scatter plots show median with interquartile range where each dot represents an individual tumour. Mann-Whitney U test was performed for statistical analyses. Full-length blots are presented in Supplementary Fig. .

Article Snippet: Rat-tail COL1 (regular and high concentration) was purchased from Discovery Labware Inc., CorningTM (Bedford, MA; Cat # 354236, regular; and # 354249, high concentration).

Techniques: Western Blot, SDS Page, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Discoidin Domain Receptors, DDR1b and DDR2, Promote Tumour Growth within Collagen but DDR1b Suppresses Experimental Lung Metastasis in HT1080 Xenografts

doi: 10.1038/s41598-020-59028-w

Figure Lengend Snippet: Impact of DDRs on YAP1 transcriptional targets in HT1080 ± DDR/+COL1 tumours. Total RNA was isolated from s.c. ±DDR1b ( A , C , E ) and ±DDR2 ( B , D , F ) tumours (n = 4 and 5 for −DDR1b and +DDR1b tumours, respectively, and n = 4 for both –DDR2 and +DDR2 tumours) and qPCR analyses for human CTGF ( A , B ), CYR61 ( C , D ) and AXL ( E , F ) were conducted as described in the Methods section. Folds of mRNA expression between ±DDR tumours are indicated.

Article Snippet: Rat-tail COL1 (regular and high concentration) was purchased from Discovery Labware Inc., CorningTM (Bedford, MA; Cat # 354236, regular; and # 354249, high concentration).

Techniques: Isolation, Expressing