Fig 2: Higher levels of Aß40 and Aß42 are found in the media of A549 cells as compared to that of H1299 cells. Cells (0.2 × 105) were grown in 10% FBS-supplemented media for 24 h then incubated in serum-free media for 24 h and treated as indicated for 72 h with 600 µM 4-MU and/or the CD44 antibody (5F12, 5 µg/mL). The media was then collected and the same amount of protein (3 µL of 600 µg/mL total protein) of each sample was used to quantitate Aß (“Methods” section) using the indicated antibodies. Data from five independent assays, each carried out in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to controls including all components but using 6E10 antibodies instead of biotin-6E10 (A,B) or 4G8 antibodies instead of biotin-4G8 (C, D) using the GraphPad 8.4.3 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks (*) indicate a statistically significant difference from the corresponding negative control of each cell line, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test. *p < 0.05, **p < 0.01.

Fig 3: HD cultures do not rely on matrix cues or N-cadherin interactions to prevent anoikis. (A) Representative micrographs of hMSC (5 × 105 cells/mL (LD), 5 × 106 cells/mL (HD)) in 1:0.375 and 1:0.75 S-HA-PEGDA hydrogels after 24 h. (B) Viability of HD cultures of hMSC encapsulated within 1:0.75 hydrogels for 24 h and treated with anti-CD44 (CD44+) antibodies or isotype controls (CD44-) and normalized to vehicle controls, and (C) in both HD and LD cultures for 72 h (n = 3, ***P < 0.001). (D) Viability of HD cultures of hMSC in 1:0.75 hydrogels for 24 h and treated with RGD sequence-containing peptides (RGD+) or scrambled peptides (RGD-) and normalized to vehicle controls, and (E) in both HD and LD for 72 h (n = 3, **P < 0.01, ***P < 0.001). (F) Representative micrographs of hMSC in LD and HD cultures for 72 h in 1:0.75 hydrogels stained with Phalloidin-TRITC. (G) Schematic highlighting theoretical distances between any two ‘nearest neighbor’ cells, where x = 69.8 µm and y = 32.4 µm. (H) Viability of LD and HD cultures of hMSC encapsulated in 1:0.75 hydrogels for 72 h and treated with anti-N-cadherin antibody (NC+) or isotype controls (NC-) and normalized to vehicle controls (n = 3). (I) Viability of HD cultures of hMSC in 1:0.375 and 1:0.75 S-HA-PEGDA hydrogels after up to 28 days (n = 3). In (A) and (F) scale bar = 100 µm. Plots show mean + SD. In (B)–(E) and (H) a two-tailed Mann-Whitney test, and in (I) a Kruskal-Wallis test followed by Dunn's Multiple Comparison were used to detect statistical significance.

Fig 4: hMSC within S-HA-PEGDA hydrogels synthesize and secrete proteins pericellularly. a Reaction scheme for hydrogel formation. Thiol-modified hyaluronic acid (S-HA) cross-links with poly(ethylene glycol) diacrylate (PEGDA) to form a hydrogel via a Michael addition. b Viability of hMSC encapsulated in 1:0.75 hydrogels treated with an anti-CD44 (CD44+) antibody or an isotype control (CD44-) for 24 h and normalized to vehicle controls (n = 3). c Viability of hMSC encapsulated in 1:0.75 hydrogels treated for 24 h with RGD sequence-containing peptides (RGD+) or scrambled peptides (RGD-) and normalized to vehicle controls (n = 3). In b and c plots show means ± s.d. d Representative micrographs showing fluorescence labeling of the methionine analogue l-homopropargylglycine (HPG, green) in hMSC-laden 1:0.375, 1:0.75, and 1:3 hydrogels after 72 h in culture. The cell membrane, as determined by differential interference contrast (DIC) imaging, is outlined in white. Scale bar = 100 µm, inset = 10 µm. Plots show mean intensity of the fluorescence signal (±s.e.m.) as a function of distance from the cell membrane in 1:0.375 (blue dashes), 1:0.75 (green dashes), and 1:3 (red dashes) hydrogels and were generated from 40 profile plots collected from 30 cells per condition. e Fraction of SILAC heavy-labeled proteins in decellularized 1:0.375 (blue circles), 1:0.75 (green circles), and 1:3 (red circles) hydrogels cultured for 72 h. Scatterplots show two technical replicates (H/L count = 3) for each hydrogel composition. Gene names for ECM proteins showing high levels (>40%) of SILAC incorporation are highlighted in each panel

Fig 5: SRGN promotes lung cancer cell stemness via Nanog induction in a CD44-depedent manner. (a) Sphere formation of H1299/Mock and h1299/SRGN cells is shown. Phase contrast images of spheres (original magnification, × 100; scale bar: 50 µm) were taken on indicated days. On day 13, percentage of sphere formation was scored and is shown in bar graph. (b) Cells were cultured in serum-free medium for 48 h, and the expression of stemness factors was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR; left panel) and western blot analyses using anti-Oct3/4 (sc-9081, Santa Cruz Biotech, Santa Cruz, CA, USA), anti-SOX2 (sc-17320, Santa Cruz Biotech) and anti-Nanog (#4903, Cell Signaling Technology, Danvers, MA, USA; right panel). (c) Sphere formation in H460 cells stably harboring scramble-shRNA and SRGN-shRNA is shown. On day 7, phase contrast images of spheres (original magnification, × 100; scale bar: 100 µm) were taken, and % of sphere formation scored. (d) Western blotting of stem factors in H460 cells cultured in serum-free medium for 48 h. (e) H1299/Mock and H1299/SRGN cells stably harboring scramble-shRNA and Nanog-shRNA were subjected to sphere formation assay. On day 13, phase contrast images of spheres (original magnification, × 100; scale bar: 50 µm) were taken and the % of sphere formation scored. Western blot analysis of Nanog is shown. (f) Unsorted and CD44(-) cells stably harboring Mock-control and SRGN-expressing vectors were cultured in serum-free medium for 48 h, and subjected to western blot analysis of Nanog expression. (g) Cells described in (f) were subjected to sphere formation assay. On day 13, phase contrast images of spheres (original magnification, × 100; scale bar: 50 µm) were taken, and the % of sphere formation scored. (h) H1299 CD44(-) cells stably harboring Mock-control and CD44s-expressing vectors were cultured in the presence of CM collected from H1299/Mock or H1299/SRGN cells for 24 h, and subjected to western blot analysis of Nanog expression. (i) Cells described in h were subjected to sphere formation assay in CM collected from H1299/Mock or H1299/SRGN cells supplemented with 1X B27, 20 ng/ml EGF and 20 ng/ml bFGF. On day 13, phase contrast images of spheres (original magnification, × 100; scale bar: 50 µm) were taken, and the percentage of sphere formation scored. (j) Cells were cultured in serum-free medium containing 2 µg/ml of mouse IgG (mIgG) or CD44 antibody (5F12) (MA5-12394, Thermo Fisher Scientific, Waltham, MA, USA) for 24 h, and subjected to western blot analysis of Nanog expression. (k) Cells were seeded at 1 × 104 cells/well in 96-well plates, and treated with cisplatin at designated concentrations for 72 h (upper panel) and with 5 µM cisplatin for designated time (lower panel), and cell viability was assessed using Cell Counting Kit-8. (l) Cells (2 × 104 cells/well) were cultured in serum-free medium in 6-well ultra-low attachment plates. After 48 h, cells were stained by trypan blue and viable cells were counted. Data are presented as the mean±s.d. of three independent experiments. **P<0.01 and ***P<0.001 by Student's t-test.