Journal: bioRxiv

Article Title: ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

doi: 10.1101/2025.07.21.666015

Figure Lengend Snippet: ROCK2 inhibition with GV101 reduces CAF-mediated remodelling of pre-existing collagen in murine and human models of TNBC, also see Figures S1 and S2 (A) Schematic of collagen contraction assays utilising CAFs isolated from end-stage MMTV-PyMT tumours. (B) Representative images of PyMT CAF matrices treated with DMSO, GV101 or Fasudil at days 3 and 12 of contraction (i) and quantification of PyMT CAF organotypic matrix size over a 12-day contraction period (ii) and on day 12 (iii). (C) Representative SHG images of PyMT CAF matrices to assess collagen I (top panel, magenta), picrosirius red staining to assess fibrillar collagen (middle panel) and polarised light birefringence imaging to assess collagen bundling and density (bottom panel). (D) Quantification of unconfined compression analysis to assess Young’s modulus. (E-G) Quantification of PyMT CAF matrices; SHG peak signal intensity (E), picrosirius red staining coverage (F) and total birefringence signal (G). (H) Schematic of collagen contraction assays utilising CAFs isolated from human TNBC patients. (I) Representative images of human CAF matrices treated with DMSO, GV101 or Fasudil at days 3 and 12 of contraction (i) and quantification of CAF organotypic matrix size over a 12-day contraction period (ii) and on day 12 (iii). (J) Representative SHG images of human CAF matrices to assess collagen I (top panel, magenta), picrosirius red staining to assess fibrillar collagen (middle panel) and polarised light birefringence imaging to assess collagen bundling and density (bottom panel). (K) Quantification of unconfined compression analysis to assess matrix Young’s modulus. (L-N) Quantification of human CAF matrices; SHG peak signal intensity (L), picrosirius red staining coverage (M) and total birefringence signal (N). Data represents mean ± SEM of three individual repeats. p-values were determined using One-sample t and Wilcoxon test for normalised data and for remaining data a One-way ANOVA with multiple comparisons was used. ns = not significant (p≥0.05), * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. Scale bar = 1cm (B and I), 100µm (C and J).

Article Snippet: Slides were then incubated in 0.02% phosphomolybdic acid for 2 minutes and briefly washed in water before a 2 hour incubation in 0.1% Picrosirius Red solution (Australian Biostain solution).

Techniques: Inhibition, Isolation, Staining, Imaging

Journal: bioRxiv

Article Title: ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

doi: 10.1101/2025.07.21.666015

Figure Lengend Snippet: (A) Schematic of cell-derived matrix (CDM) production assays where both PyMT and human CAFs are treated with ascorbic acid to stimulate matrix production in the presence of DMSO, GV101 or Fasudil. (B) Representative images of PyMT CAF derived CDMs showing SHG imaging (top panel, magenta) and picrosirius red stained matrices (bottom panel). (C) Quantification of peak SHG signal (i) and picrosirius red staining coverage for PyMT CAF generated CDMs (ii). (D) Representative images of human CAF derived CDMs showing SHG imaging (top panel, magenta) and picrosirius red stained matrices (bottom panel). (E) Quantification of peak SHG signal (i) and picrosirius red coverage (ii) for human CAF generated CDMs. Data represents mean ± SEM of three individual repeats. p-values were determined using One-sample t and Wilcoxon test for normalised data and for remaining data a One-way ANOVA with multiple comparisons was used. ns = not significant (p≥0.05), * = p<0.05, ** = p<0.01. Scale bar = 100μm.

Article Snippet: Slides were then incubated in 0.02% phosphomolybdic acid for 2 minutes and briefly washed in water before a 2 hour incubation in 0.1% Picrosirius Red solution (Australian Biostain solution).

Techniques: Derivative Assay, Imaging, Staining, Generated

Journal: Bio-protocol

Article Title: Cell-derived Matrix Assays to Assess Extracellular Matrix Architecture and Track Cell Movement

doi: 10.21769/BioProtoc.4570

Figure Lengend Snippet: Examples with intact fibroblasts, including picrosirius red staining (a.) imaged using bright field microscopy (top left), binary overlay (bottom left), and by polarized light (birefringence, top right) to assess collagen I and III abundance and orientation (bottom right). Second-harmonic generation (SHG) (b.) imaging (top) and analysis of fiber orientation (bottom). Applications of CDMs with fibroblast removed and cancer cells seeded on top to assess single cancer cell migration (left) and collective migration (right).

Article Snippet: 3 Picrosirius red staining kit (Australian Biostain, APSRA, 500 mL) .

Techniques: Staining, Microscopy, Imaging, Migration

Journal: EBioMedicine

Article Title: SPARC dependent collagen deposition and gemcitabine delivery in a genetically engineered mouse model of pancreas cancer

doi: 10.1016/j.ebiom.2019.09.024

Figure Lengend Snippet: A: PDAC frequency in KC- SPARC WT ( n = 29, 65% tumour frequency) and KC- SPARC −/− ( n = 53; 49% tumour frequency, p = .1, Fishers Exact test). B: Survival analysis of tumour bearing KC- SPARC WT ( n = 16) and KC- SPARC −/− ( n = 25) mice shows significantly reduced survival of KC-SPARC −/− mice (280 days versus 485 days, p = .02, log-rank-test). C: Masson trichrome (MT) and picrosirius red staining in pancreatic tumours derived from KC- SPARC WT and KC- SPARC −/− mice shows reduced overall collagen, in particular collagen I and III (picrosirius red). D: Automated quantification of MT in tumours from KC- SPARC WT ( n = 8) and KC- SPARC −/− ( n = 17) mice ( p < .05, Mann Whitney U test) E: Automated quantification of picrosirius red in tumours from KC- SPARC WT ( n = 7) and KC- SPARC −/− ( n = 18) mice. Collagen is significantly reduced in KC-SPARC −/− mice ( p = .01; Mann Whitney U test). F: Automated quantification of α-SMA immunohistochemistry in tumours from KC- SPARC WT ( n = 8) and KC- SPARC −/− ( n = 16) mice ( p = .7, Mann-Whitney-U test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following reagents were used for special extracellular matrix stains: Picrosirius Red Stain Kit (Polysciences Inc., Cat. 24901-250), Masson trichrome staining kit (Polysiences Inc., Cat. 25088-1), Weigert's iron hematoxylin kit (Sigma) and hyaluronic acid binding protein (Calbiochem, Cat. 385911).

Techniques: Staining, Derivative Assay, MANN-WHITNEY, Immunohistochemistry

Journal: Science Advances

Article Title: Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response

doi: 10.1126/sciadv.adl1197

Figure Lengend Snippet: ( A ) Representative images of nonneoplastic pancreas, early-, mid- and late-stage KPC tumors stained for hematoxylin and eosin (H&E) (top row), Picrosirius Red imaged by bright-field (second row) and polarized light (third row; matched to Picrosirius Red images), and SHG imaging (bottom row). Scale bars, 100 μm (H&E and Picrosirius Red bright-field and polarized light) and 25 μm (SHG). ( B ) Representative dark-field images of decellularized WT pancreas (top left) and late-stage KPC specimen (top right). Representative image of collagen IV IF (green) of decellularized WT pancreas (bottom). Scale bars, 500 μm (dark-field) and 100 μm (collagen IV). ( C ) Workflow for matrisome-enriched DIA LC-MS/MS analysis of WT, KP fl C, and KPC tumors at early-, mid-, and late-stage time points. m / z , mass/charge ratio; 0.5% SDC, sodium deoxycholate. ( D ) Classification of matrisome categories detected . Core matrisome ( n = 123; purple) and matrisome-associated ( n = 158; gray). Core matrisome category (purple colors); ECM glycoproteins ( n = 83), collagens ( n = 27), and proteoglycans ( n = 13). Matrisome-associated category (gray colors); ECM regulators ( n = 93), ECM-affiliated proteins ( n = 46), and secreted factors ( n = 19). ( E ) Principal components analysis of WT (green), KP fl C (blue), and KPC (red) tumors at early- (circle), mid- (star), and late-stage (square) time points. ( F ) Volcano plot of matrisome proteins comparing KP fl C and KPC mid-stage tumors. Two-sample t test with FDR = 0.05. y axis = −log( P value) and x axis = difference (fold change). ITIH3, Inter-Alpha-Trypsin Inhibitor Heavy Chain 3; LAMC1, Laminin Subunit Gamma 1; ADAM10, A Disintegrin And Metalloproteinase Domain 10; PLXNB2, Plexin B2; S100A16, S100 Calcium Binding Protein A16; MUC2, Mucin 2; PRSS1, Serine Protease 1; SERPINA3K, serine (or cysteine) peptidase inhibitor, clade A, member 3K; ECM1, Extracellular Matrix Protein 1; COL7A1, Collagen Type VII Alpha 1 Chain. Schematics were created with Biorender.com .

Article Snippet: Slides were dewaxed using the Leica ST5010 Autostainer XL and then stained manually with 0.02% phosphomolybdic acid and 0.1% Picrosirius Red (Australian Biostain) for fibrillar collagens.

Techniques: Staining, Imaging, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Science Advances

Article Title: Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response

doi: 10.1126/sciadv.adl1197

Figure Lengend Snippet: ( A ) 3D organotypic contraction assay with CRISPRi CAFs. ( B ) Representative ROIs of NID2 IHC with NID2-positive cell (%) quantification. One-way ANOVA with Dunnett’s test, * P < 0.05 and ** P < 0.01. n = 3 repeats, in triplicate. Scale bars, 50 μm. ( C ) Quantification at days 3, 6, 9, and 12 and representative images of matrix contraction (in square millimeter) at days 3 and 12. Scale bars, 1 cm. ( D ) Young’s modulus (in kilopascals) of matrices on day 12. One-way ANOVA with Dunnett’s test, ** P < 0.01. n = 3 repeats, in triplicate. ( E ) Representative SHG maximum intensity with quantification. One-way ANOVA with Dunnett’s test, * P < 0.05. n = 3 repeats, in triplicate. Scale bars, 100 μm. ( F ) Representative ROIs of Picrosirius Red with coverage (%) quantification. One-way ANOVA with Dunnett’s test, * P < 0.05 and ** P < 0.01. n = 3 repeats, in triplicate. Scale bars, 100 μm. ( G ) Representative ROIs of polarized light Picrosirius Red (matched to Picrosirius Red ROIs) with coverage (%) quantification. One-way ANOVA with Dunnett’s test; green, yellow, and red-orange birefringence proportion quantification. Two-way ANOVA with Dunnett’s test, * P < 0.05 and ** P < 0.01. n = 3 repeats, in triplicate. Scale bars, 100 μm. ( H ) Organotypic invasion assay. Representative H&E ROIs of cancer cell invasion with quantification. One-way ANOVA with Dunnett’s test, *** P < 0.001. n = 5 GFP-1 KRAB, n = 7 B500 NID2 KRAB, and n = 8 C500 NID2 KRAB repeats, in triplicate. Scale bars, 100 μm. All data represented as means ± SEM. Schematics were created with Biorender.com .

Article Snippet: Slides were dewaxed using the Leica ST5010 Autostainer XL and then stained manually with 0.02% phosphomolybdic acid and 0.1% Picrosirius Red (Australian Biostain) for fibrillar collagens.

Techniques: Contraction Assay, Invasion Assay

Journal: Science Advances

Article Title: Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response

doi: 10.1126/sciadv.adl1197

Figure Lengend Snippet: ( A ) Subcutaneous coinjection experiment using GFP-1 KRAB or B500 NID2 KRAB CAFs (75%) with cancer cells (25%). Mice were treated twice weekly with gemcitabine/Abraxane, beginning day 11. ( B ) Quantification of GFP-1 KRAB (purple) and B500 NID2 KRAB (blue) tumor growth (in cubic millimeters) over time and at day 23. Welch’s t test, * P < 0.05. n = 9 GFP-1 KRAB mice and n = 7 B500 NID2 KRAB mice. IP, intraperitoneal injection. ( C ) Representative images of day 23 tumors. Scale bars, 2 cm. ( D ) Representative ROIs of NID2 IHC with quantification of positive pixels (%). Mann-Whitney test, * P < 0.05. Scale bars, 100 μm. ( E ) Representative ROIs of NID1 IHC with quantification of positive pixels (%). Welch’s t test, ns: P > 0.05. Scale bars, 100 μm. ( F ) Representative ROIs of Picrosirius Red with quantification of coverage (%). Welch’s t test, * P < 0.05. Scale bars, 100 μm. ( G ) Representative ROIs of polarized light Picrosirius Red with quantification of coverage (%). Welch’s t test, ** P < 0.01. Quantification of proportions of green, yellow, and red-orange birefringence. Two-way ANOVA with Sidak’s test, *** P < 0.001 for green and yellow birefringence. Scale bars, 100 μm. ( H ) Representative ROIs of CD31 IHC with quantification of positive pixels (%). Welch’s t test, *** P < 0.001. Scale bars, 100 μm. All data represented as means ± SEM. Schematics were created with Biorender.com .

Article Snippet: Slides were dewaxed using the Leica ST5010 Autostainer XL and then stained manually with 0.02% phosphomolybdic acid and 0.1% Picrosirius Red (Australian Biostain) for fibrillar collagens.

Techniques: Injection, MANN-WHITNEY

Journal: Science Advances

Article Title: Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response

doi: 10.1126/sciadv.adl1197

Figure Lengend Snippet: ( A ) Orthotopic coinjection experiment using GFP-1 KRAB or B500 NID2 KRAB CAFs (75%) with luciferase cancer cells (25%). Vehicle mice culled day 44 ( n = 8 GFP-1 KRAB and n = 6 B500 NID2 KRAB). Chemotherapy mice culled day 65 ( n = 7 GFP-1 KRAB and n = 7 B500 NID2 KRAB). ( B ) IVIS signal (day 7) for GFP-1 KRAB and B500 NID2 KRAB mice with Fstop1, exp30s. Color scale is radiance. ( C ) Pancreatic tumor weight (in grams). Welch’s t test, ns: P > 0.05. ( D ) Number of visible liver metastases. Welch’s t test, * P < 0.05. ( E ) Representative livers at end point. Scale bars, 1 cm. ( F ) Liver metastasis coverage. Welch’s t test, *** P < 0.001. ( G ) H&E liver sections (top) and QuPath detection (bottom). Scale bars, 5 mm. ( H ) Representative ROIs of Picrosirius Red with coverage (%) quantification. Welch’s t test, * P < 0.05. Scale bars, 100 μm. ( I ) Representative ROIs of polarized light Picrosirius Red with coverage (%) quantification. Welch’s t test, *** P < 0.001. Scale bars, 100 μm. ( J ) Pancreatic tumor weight (in grams). Welch’s t test, ** P < 0.01. ( K ) Number of visible liver metastases. Welch’s t test, * P < 0.05. ( L ) Representative livers at end point. Scale bars, 1 cm. ( M ) Liver metastasis coverage. Mann-Whitney test, ** P < 0.01. ( N ) H&E liver sections (top) and QuPath detection (bottom). Scale bars, 5 mm. ( O ) Representative ROIs of Picrosirius Red with coverage (%) quantification. Welch’s t test, **P < 0.01. Scale bars, 100 μm. ( P ) Representative ROIs of polarized light Picrosirius Red with coverage (%) quantification. Welch’s t test, * P < 0.05. Scale bars, 100 μm. Schematics were created with Biorender.com .

Article Snippet: Slides were dewaxed using the Leica ST5010 Autostainer XL and then stained manually with 0.02% phosphomolybdic acid and 0.1% Picrosirius Red (Australian Biostain) for fibrillar collagens.

Techniques: Luciferase, MANN-WHITNEY