Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: (A) Schematic representation of SLC3A2-containing amino acid transporters. (B) MDA-MB-231 and (C) PANC1 cells were seeded on 2 mg/ml collagen I (Coll) or plastic (P) under complete or Glc starvation media for 24 hours, mRNA was extracted and the levels of SLC3A2, SLC7A5, SLC7A6, SLC7A8 and GAPDH as housekeeping gene were quantified by SYBR-green qPCR. Data are presented as 2 -ΔCT ; mean ± SEM, N=3 independent experiments, ****p < 0.0001 Kruskal-Wallis, Dunn’s multiple comparisons test. (D,E) MDA-MB-231 and (F,G) PANC1 cells were seeded on 2 mg/ml collagen I (Coll) or plastic (P) under Glc starvation for one or two days, respectively. Cells were fixed and stained with (D,F) Hoechst 33342 (magenta), SLC3A2 (cyan) and Phalloidin Alexa Fluor 555 (grey) or (E,G) Hoechst 33342 (cyan), SLC7A5 (red) and Phalloidin Alexa Fluor 555 (grey). Bar, 20 µm. Images were collected with a ZEISS LSM98O Airyscan2 microscope and analysed by Fiji/lmageJ software. Data are presented as mean ± SEM, N=3 independent experiments (the bigger dots represent mean intensity of each field of view); **p < 0.01, ***p < 0.001 Mann-Whitney test. (H, I) MDA-MB-231 and PANC1 cells were transfected with an siRNA targeting SLC3A2 (3A2), an siRNA targeting SLC7A5 (7A5) or a combination of siRNA targeting SLC3A2 and SLC7A5 (3A2+7A5) and grown on 2mg/ml collagen I under Glc starvation for one or three days, respectively. Metabolites were extracted and analysed by targeted mass spectrometry. N=3 independent experiments.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: SYBR Green Assay, Staining, Microscopy, Software, MANN-WHITNEY, Transfection, Mass Spectrometry

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: MDA-MB-231 (A-E) and PANC1 (F-O) cells were transfected with an siRNA targeting SLC3A2 (3A2 si), an siRNA targeting α2 integrin (α2 si) or a non-targeting siRNA control (Nt si) and grown on 2 mg/ml collagen I in Glc starvation media for one (A-E), two (F-J) and three days (K-O). Protein level of phosphorylated S6 (pS6, B,G,L), LC3II (C,H,M), SLC3A2 (D,I,N) and α2 integrin (E,J,O) were measured via Western blotting. Data are presented as mean ± SEM, N≥4 independent experiments, *p < 0.05, **p < 0.01 Kruskal-Wallis, Dunn’s multiple comparisons test.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: MDA-MB-231 (A-D) arid PANC1 (E-H) were transfected with an siRNA targeting SLC3A2 (3A2 si), an siRNA targeting SLC7A5 (7A5 si) or a non-targeting siRNA control (Nt si) and grown on 2 mg/ml collagen I under complete or Glc starvation media for 4 days. Cells were fixed and stained with Hoechst 33342. Images were collected by an ImageXpress micro and analysed by Meta×press software. Data are presented as mean ± SEM, N=3 independent experiments, *p < 0.05, **p < 0.01 and ****p < 0.0001 Mann-Whitney test. (I) MDA-MB-231-GFP cells were transfected with a combination of siRNAs targeting SLC3A2 and SLC7A5 (3A2+7A5 si) or a non­targeting siRNA control (Nt si) for 24 hours, spheroids were generated by the hanging drop method and embedded in 3 mg/ml collagen I and Geltrex (50:50 ratio) for 3 days under Glc starvation. Images were collected by a Nikon Confocal A1 microscope. Invasion area was quantified with Fiji/lmageJ. Bar, 200 µm. Data are presented as mean ± SEM, N=2 independent experiments. (J) PANC1 cells were transfected as in I, spheroids were generated by the hanging drop method and embedded in 3 mg/ml collagen land Geltrex (50:50 ratio) for 6 days under Glc starvation. Live images were collected every day by an Olympus E45O microscope. Bar, 250 µm. Data are presented as mean ± SEM, N=2 independent experiments. (K, L) E0771 mouse tumours organoids were grown in a 3 mg/ml collagen I and Geltrex (50:50) mixture and starved in Glc starvation media for 2 days in the presence or absence of 50 mM D-phenylalanine (D-Phe). Spheroids were imaged live every day by an Olympus E45O microscope. Bar, 250 µm. Data are presented as mean ± SEM, N=3 independent experiments, *p < 0.05, **p < 0.01 and ****p < 0.0001; (l,J,K) Two-way ANOVA, Tukey’s multiple comparisons test; (L) Mann-Whitney test.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Transfection, Control, Staining, Software, MANN-WHITNEY, Generated, Microscopy

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: (A,C) RNA sequencing data from basal-Like breast tumours (n=135) and normal breast tissue (n=291) or pancreatic adenocarcinoma tumours (n=179) and normal pancreatic tissue (n=171) for the co-expression of SLC3A2 and SLC7A5. *p< 0.05. Data were obtained from gepia2.com. (B) Overall survival of basal-like breast cancer patients with high (red) or low (blue) co­expression of SLC3A2 and SLC7A5. Data were obtained from gepia2.com. (D) Disease free survival of pancreatic adenocarcinoma patients with high (red) or low (blue) co-expression of SLC3A2 and SLC7A5. Data were obtained from gepia2.com. (E,F) RNA sequencing data and ROC analysis for the co-expression of SLC3A2 and SLC7A5 from chemoresistant (Non-responder) and chemosensitive (Responder) breast tumours. (E) p = 1,4e-6, Mann-Whitney test. (D) AUG = 0.628; ROC p value = 2.5e-07. Data were generated in ROCplot.com.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: RNA Sequencing, Expressing, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: Under Glc starvation, collagen I promoted α2β1 integrin-dependent mTORCI activation in breast and pancreatic cancer cells. Collagen I also increased the membrane levels of the LAT1-4F2hc amino acid transporter heavy chain, SLC3A2 (3A2), and light chain, SLC7A5 (7A5), stimulating amino acid import. This resulted in mTORCI activation and autophagy inhibition, leading to increased survival, proliferation and invasion, in 2D and 3C contexts.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Activation Assay, Membrane, Inhibition