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1 Soft fibrin gels promote selection and growth of tumorigenic cells Jing Liu $, Youhua Tan $, Huafeng Zhang, Yi Zhang, Pingwei Xu, Junwei Chen, Yeh-Chuin Poh, Ke Tang, Ning Wang*, Bo Huang* $ These authors contributed equally. *Correspondence should be addressed to: Ning Wang (nwangrw@illinois.edu) or Bo Huang (tjhuangbo@hotmail.com) List of Contents 1. Additional methods (page 2-5) 2. Table S1 and S2, Figure S1-S22 with legends (page 6-29) NATURE MATERIALS 1

2 1. Additional methods Conventional 2D rigid dish and 3D fibrin gel cell culture of tumour cells Before seeded into 3D fibrin gels, B16-F1 cells were maintained in the standard culture conditions as mentioned above. After 0.25% trypsin treatment (Sigma-Aldrich, St. Louis, MO), cells were detached and suspended in MEM (10% FBS) and cell density was adjusted to 10 4 cells/ml. Fibrinogen was diluted into 2 mg/ml with T7 buffer (ph 7.4, 50 mm Tris, 150 mm NaCl). 1:1 fibrinogen and cell solution mixture was made by mixing the same volume of the fibrinogen solution and the cell solution, resulting in 1 mg/ml fibrinogen and 5000 cells/ml in the mixture. 250 µl cell/fibrinogen mixtures was seeded into each well of 24 well-plate and mixed well with pre-added 5 µl thrombin (0.1 U/µl). The cell culture plate was then moved into 37 C cell culture incubator and incubated for 10 min. Finally, 1 ml MEM medium containing 10% FBS and antibiotics were added. Similar procedures were applied to EL4, H22 cells, HepG2, A2780 and P815 cells, which were cultured in RPMI-1640 cell culture medium (Invitrogen) with 10% FBS. 2D fibrin gels were prepared following the same protocol of 3D fibrin gels but without premixing cells with fibrinogen solution. After the gels were polymerized, B16-F1 cells were seeded on top of fibrin gels. α v β 3 blocking antibody crgdfv (Enzo Life Sciences), β 1 blocking antibody (Abcam Inc), ROCK inhibitor Y (Calbiochem) were purchased. All drugs were pre-diluted in normal culture medium to the indicated concentrations and then used for cell treatment. Different concentrations of doxorubicin or cisplatin were added to the B16-F1 cell culture medium during the last 18 hours of 5-day culture in 90-Pa 3D fibrin gels or conventional 2D rigid dish. Cells were collected and stained with FITC conjugated Annexin-V for apoptotic detection by flow cytometry. The result presented was the apoptosis% calculated by Annexin V positive cell number divided by total cell number. To determine if the data in Fig. 3e is due to poor accessibility to the drug for the cells in the 3D fibrin gels, we added the same concentration of doxorubicin to the culture media of 3D fibrin gels and 2D rigid plastic. We harvested cells at different time points (4th and 8th hour) and analyzed the cells by flow cytometry, and found the similar mean fluorescent intensity (doxorubicin exhibits red fluorescence) and positive cell ratio between two groups, suggesting that the drug entered a similar number of cells both in 3D fibrin gels and on 2D surface. Therefore what we found is not due to limitation of drug accessibility in the 3D fibrin gels. The fresh breast cancer tissue removed from patients was digested with collagenase and hyaluronidase for 1 h. After grinding with semi-frosted slides and lysis of RBC, the dissociated cells were incubated on ice for 20 min and then spun down at 500 rpm for 1 min. This process was repeated twice and the cells were first incubated for 2 h to get rid of adhesive cells (e.g., macrophages; within 2 hr, tumour cells could not adhere to the plate) and then used as the primary breast cancer cells. Peripheral blood cells of leukemic patients were collected and separated by the Ficoll gradient centrifugation. The obtained peripheral blood mononuclear cells were used for T- leukemic cell purification by negative magnetic sorting with biotinylated anti-human CD14, CD16, CD19, CD36, CD56, CD123 and CD235a antibodies (Pan T-Cell Isolation Kit II, 2 NATURE MATERIALS

3 SUPPLEMENTARY INFORMATION Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated primary T-leukemic cells were cultured in 3D fibrin gels (90-Pa). Serial transplantation assay We subcutaneously injected 100 B16 cells derived from 3D soft fibrin gels (90-Pa) to C57BL/6 mice. After tumour formation (5x5 mm in size), we isolated B16 cells from the tumours and injected 10 5 isolated cells to naïve C57BL/6 mice. After 10 days, the tumour formation was observed in all mice (n=6). Similarly, tumour cells were isolated again and injected into 6 mice at 10 5 cells. All mice (n=6) formed tumours. Cell proliferation assay BrdU assay was used to quantify proliferating B16 cells cultured within 3D fibrin gels. Briefly, we added 10 µm BrdU (BD Biosciences) 17 hr before Day 5 and the cells were fixed 17 hr later, treated, and stained, following manufacturer s instruction. Then, 10 µg/ml propidium iodide was incubated with cells for 1 hr at room temperature. The proliferating cell number was quantified for each round colony at the equatorial plane of the spheroid. Apoptosis assay Propidium iodide (PI) was used to label apoptotic cells cultured in 3D fibrin gels with different stiffness from Day 1 to Day 5. Briefly, the cells cultured in 3D fibrin gels were rinsed with prewarmed PBS and then incubated with 10 µg/ml PI (pre-diluted in normal culture medium) for 1 h. The PI-positive (apoptotic) cells were counted under a fluorescence microscope. Cytoskeleton staining B16-F1 cell spheroids maintained in 3-D fibrin gel were used for F-actin staining. At Day 5, multicellular B16-F1 tumour spheroids were pipetted into single cells, seeded onto glassbottomed 35-mm dishes pre-coated with collagen-1 (200 g/ml), and cultured for 6 h at 37 C and5% CO 2. The cells were washed three times with PBS, and fixed with 3.7% paraformaldehyde at room temperature for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. F-actin staining of cells was performed by incubating cells with 50 µg/ml TRITC-labeled phalloidin (Sigma-Aldrich) in PBS for 20 min at room temperature. For nuclear imaging, cells were then re-stained with 1 µg/ml Hoechst for 10 min. RT-PCR and real-time PCR analysis The primers were designed with the Oligo Primer Analysis 4.0 software, and the sequences were blasted ( Total RNA (100 ng) was used for reverse transcription using Superscript II RNase H reverse transcriptase (Invitrogen) in a volume of 20 μl. Then, 1 μl of cdna was amplified with FastStart Universal SYBR Green Master (Rox) (Roche, Indianapolis, IN) in duplicate. Histologic evaluation and Immunohistochemistry The 4% paraformaldehyde fixed BALB/c mice lung tissues were embedded in paraffin and cut to 6-µm thick sections by Leica RM2016 Machine. Tissue-sections were stained by Hematoxylin NATURE MATERIALS 3

4 and Eosin (H&E) and slides were evaluated for inflammation and tumour formation by a veterinary pathologist blinded to sample identity. Immunohistochemistry detecting fibrinogen (1:500; DAKO, Glostrup, Denmark) was performed on paraffin-embedded sections using the Vectastain ABC kit for rabbit primary antibodies (Vector Labs, Burlingame, CA), following manufacturer s instructions. EGF (epidermal growth factor) and AKT1/2 kinase inhibitor were purchased from Sigma-Aldrich; anti-phosphorylated AKT1/2 antibody was obtained from Millipore. Quantification of cell stiffness The complex stiffness was defined as G* = /d, where d was the peak amplitude of the bead displacement. To increase the signal to noise ratio, d was averaged over 5 consecutive cycles. The measured stiffness has the unit of Pa/nm. The magnetic bead was embedded ~50% into the cell surface. A finite element model was used to convert cell stiffness (Pa/nm) to shear modulus (Pa) 1. Since cell stiffness is defined as the ratio of applied stress to cell deformation (cell shape change or cell strain) and cell deformability is defined as the degree of cell deformation (or the degree of cell shape change) under a given level of applied stress, cell stiffness is the inverse of cell deformation or cell deformability. Transfection assay Sox2, c-kit, nestin, and Bmi-1 sirnas and the corresponding scrambled control oligonucleotides were purchased from RiboBio Corporation (Guangzhou, China). Briefly, 100 nm sirna each was transfected to the cells cultured in 24-well plate by Lipofectamine 2000 (Invitrogen), according to instructions provided by the manufacturer. Assay of tumour cell arrest in lung B16-F1 cells were cultured on rigid plastic or in 3D soft fibrin gels (90 Pa) for 5 days. They were then labeled with CFSE and injected into mice ( per mouse) via tail vein. Lungs were surgically excised from mice 4 h, 12 h and 24 h after the injection. Frozen sections were prepared and analyzed by fluorescence microscopy. Fluorescent spots, which represented single cells, were counted from 20 randomly chosen viewing fields in the sections of each mouse. Traction measurement In brief, red fluorescent beads (0.2 µm in diameter) were embedded into the apical surface of gels. Images of these beads under a cell were taken before and after trypsinization. Based on an established method of traction force computation, the displacements of the beads were recorded, from which the traction field was then computed with the known gel modulus 2. The substrate stiffness of the gel was varied by altering bis-acrylamide crosslinker concentrations (0.04, 0.06, 0.3%) and polyacrylamide concentration (3%, 3%, 5%) and the corresponding stiffnesses are 0.15, 0.6, and 8.0 kpa respectively using published protocols 2,3. Cell transmigration assays 4 NATURE MATERIALS

5 SUPPLEMENTARY INFORMATION The cell transmembrane migration assay has been described in detail previously 4. 8-µm-pore transwell migration chambers were purchased from Costar. The membranes were pre-coated at room temperature for 1 hour with rat collagen-i (66 g/ml) (Sigma). 5% FBS-MEM was added in the lower chamber as a chemoattractant. Cells (1.5x 10 4 per well) were added to the upper chambers in serum-free medium. Cell migration was allowed to proceed for 3, 6 or 9 hours at 37 in CO 2 incubator. Cells then were washed with PBS and fixed in methanol. The cells from the upper surface were removed with a cotton swab. Cells that migrated to the lower surface were stained with crystal violet and counted. At least 10 random viewfields were counted for each experimental condition. Curve fitting The data in Fig. 1 (c) and (d) were fitted by the 3 rd order polynomial functions (f(x) = Ax 3 +Bx 2 +Cx+D). The parameters of the fitting curves (A, B, C, and D) are ( , , and ), (-5.787, , and ), and ( , 9.504, and ) in (c); and (3954.5, , and ), (1888.1, , and ), and (780.47, , and ) in (d) for 90-Pa, 420-Pa and 1050-Pa fibrin gels, respectively. A two-tailed Student s t-test was used for all statistics. References 1. Mijailovich, S.M., Kojic, M., Zivkovic, M., Fabry, B. & Fredberg J.J. A finite element model of cell deformation during magnetic bead twisting. J. Appl. Physiol. 93, (2002). 2. Chowdhury, F. et al. Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells. Nat. Mater. 9, (2010). 3. Poh, Y.C., Chowdhury, F., Tanaka, T.S. & Wang. N. Embryonic stem cells do not stiffen on rigid substrates. Biophys. J. 99, L01-L03 (2010). 4. Senger, D.R. et al. The alpha(1)beta(1) and alpha(2)beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis. Am J Pathol. 160, (2002). NATURE MATERIALS 5

6 Table S1 Tumourigenicity of H22 and A2780 cell spheroids in different mouse models i.m.: intramuscular injection; i.p.: intraperitoneal injection. 6 NATURE MATERIALS

7 SUPPLEMENTARY INFORMATION Table S2 Primer sequences used for RT-PCR and real-time PCR analysis of B16-F1 cells NATURE MATERIALS 7

8 Fig. S1. Apoptotic colonies increase with stiffness of fibrin gels and with culture time single cells were cultured within the 3D fibrin gels and propidium iodide was used to stain the apoptotic cells from Day 1 to Day 5. Apoptotic colonies as % of total colonies (round and nonround) were plotted. The increase from Day 2 to Day 5 in apoptotic colonies (%) for 90-Pa gels could be due to additional apoptosis of non-round colonies. Mean+s.e.m.; n=6 dishes from 3 independent experiments. From Day 1 to Day 5, p=0.0003, 0.015, 0.007, 0.003, and between 90-Pa and 420-Pa gels; p=0.0003, 0.004, 0.002, , and between 90-Pa and 1050-Pa gels. There are no significant differences between 420-Pa and 1050-Pa gels (p>0.55) except at Day 2 (p<0.016). 8 NATURE MATERIALS

9 SUPPLEMENTARY INFORMATION Fig. S2. B16 cells proliferate better in softer fibrin gels than in stiffer ones. B16 cells were cultured within fibrin gels with different stiffness (90, 420, or 1050 Pa). (a) Representative images of B16 spheroids (top left panels, bright field images) in different fibrin gels were stained with anti-brdu antibody (top right panels; Brdu, green) and propidium iodide (bottom left panels; Nuc, red). Brdu was added 17 hr before Day 5 to quantitate cells that went through DNA synthesis. Bottom right panels were merged images of Brdu and Nuc. (b) Quantification of proliferating cells in one colony per focal plane. Mean+s.e.m.; n=3 independent experiments; ***p< NATURE MATERIALS 9

10 Fig. S3. Cell viability of 3D fibrin gel cultured B16 spheroid cells. (a) Trypan blue staining of 3D B16-F1 spheroid cells. B16-F1 spheroids were picked out from soft 3D fibrin gel and cell suspension was stained with 0.4% trypan blue. (b) Annexin V staining of 3D B16-F1 spheroid cells for apoptosis assay and analyzed by flow cytometry. Rigid B16-F1: control cells plated on rigid plastic dishes. 3D B16-F1: cells cultured within 90-Pa 3D fibrin gels. Positive Cont.: cells treated with cisplatin (100 M for 20 h, a known apoptosis-inducing drug). The results shown were a representative of three independent experiments. 10 NATURE MATERIALS

11 SUPPLEMENTARY INFORMATION Fig. S4. Serial passages from 3D soft fibrin gels elevate colony number. B16-F1 cells were seeded within 90-Pa (a) or 420-Pa (b) fibrin gels. 1 st generation: cells were cultured up to Day 5 and colony numbers were counted from Day 1 to Day 5. 2 nd generation: the cells from 1 st generation were cultured for another 5 days. 3 rd generation: the cells from 2 nd generation were cultured for another 5 days. For (a), between 2 nd and1 st generation, p<0.02 at Day 2-5 except at Day 1 where p>0.45. Between 3 rd and 1 st generation, p<0.025 at Day 1-5. Between 3 rd and 2 st generation, p<0.05 at Day 1-5. For (b), between 2 nd and 1 st generation, p< at Day 1-5. Between 3 rd and 1 st generation, all p< Between 3 rd and 2 st generation, p<0.03 at Day 1, 2, 4, or 5 except at Day 3 where p>0.19. Data are averaged from two independent experiments. Mean+s.e.m. NATURE MATERIALS 11

12 Fig. S5. Colony size increases more with passage number in softer 3D fibrin gels. Colony sizes are counted for the cells in 90-Pa (a) or 420-Pa (b) fibrin gels. Note that the cells were only grown up to Day 5, not Day 6 as in Fig. 1d. For (a), all p-values (see Fig. S4 for different comparison groups) are less than 0.03 except between 3 rd and 2 st generation at Day 4 and Day 5, where p>0.8. For (b), all p-values are less than except between 3 rd and 1 st generation at Day 3 and Day 4 where p>0.06. n>40 colonies were counted for each time point. 12 NATURE MATERIALS

13 SUPPLEMENTARY INFORMATION Fig. S6. B16-F1 cells grow better in 3D soft fibrin gels than in 3D soft collagen gels. Representative images of B16-F1 tumour spheroid are shown after different days in culture in 3D collagen-1 gels or fibrin gels. Collagen-1 gels: 0.6 mg/ml = 94 Pa; 1 mg/ml =160 Pa. Fibrin gel: 1 mg/ml = 90 Pa. Note that the multicellular tumour spheroid is much bigger in the fibrin gel than in collagen-1 gels at Day 5 (scale bars are different). NATURE MATERIALS 13

14 Fig. S7. 3D soft fibrin gels are better than 3D soft collagen gels in growth of tumour spheroids. Quantitation of tumour spheroid size grown in 3D fibrin gels or in 3D collagen gels as a function of culture time shows that the colony sizes in 3D collagen gels are much smaller than in 3D fibrin gels, suggesting 3D fibrin gels are better at promoting cell proliferation. 14 NATURE MATERIALS

15 SUPPLEMENTARY INFORMATION Fig. S8. 3D soft fibrin gels promote formation of tumour spheroids from various mouse and human cancer cell lines and human primary cancer cells. Images are multicellular tumour spheroids formed by A2780, EL4, H22, HepG2, and P815 cells in 3D soft fibrin gel at Day 6, respectively; primary cancer cells from a human breast cancer patient and from a leukemia patient formed smaller tumour spheroids at Day 10 in 3D soft fibrin gels (90-Pa). All cell images were captured under 10 objective lens of Leica optical microscope. NATURE MATERIALS 15

16 Fig. S9. B16-F1 cells grow better within 3D fibrin gels than on 2D fibrin gels. (a) Round colony number as a function of culture time. p<0.0002, 0.005, between 90-Pa 3D and 2D gels at Day 1, 2, and 3, respectively; no significant differences at Day 4 and Day 5 (p>0.12). All p< between 420-Pa 3D and 2D gels. There are no significant differences (p>0.05) between 1050-Pa 3D and 2D gels except at Day 3 (p<0.022). (b) Size of round colonies as a function of culture time. B16 colony sizes are larger in 3D fibrin gels than on 2D fibrin gels at Day 4 (all p<0.018) and Day 5 (all p< ) between 90-Pa 3D and 2D gels, between 420-Pa 3D and 2D gels, and between 1050-Pa 3D and 2D gels. Mean+s.e.m.; n=6 independent experiments for 3D fibrin gels and n=3 independent experiments for 2D fibrin gels. Note that for more accurate comparison, colony projected area rather than colony volume is presented in (b). Note that on 2D gels, the colony area reached a plateau between Day 4 and Day 5, whereas within 3D gels, it continued to increase. Therefore, the difference in colony size cannot be explained by the presence of fibrin polymers inside each colony in 3D gels. 16 NATURE MATERIALS

17 SUPPLEMENTARY INFORMATION Fig. S10. B16-F1 cells form small colonies on 2D soft collagen-coated polyacrylamide gels. B16-F1 cells were seeded onto both soft (0.15 kpa; left image) and stiff 2D (8 kpa; middle image) type-1 collagen-coated polyacrylamide gels, respectively, for 5 days. Cells cultured on collagen- 1 coated rigid glass dish (right image) were controls. It appears that B16-F1 spread on stiff or rigid substrates, but stay round and form small colonies on 2D soft substrates. NATURE MATERIALS 17

18 Fig. S11. Cells from 3D soft fibrin gels survive better in the lung than control cancer cells. B16-F1 cells cultured in 3D soft fibrin gels (triangles) or on the rigid plastic (squares) were labeled with CFSE and labeled cells were injected into mice via tail vein. The arrest of B16 cell in the lungs was analyzed by counting fluorescent spots in the sections prepared from mice (n=3) at different time points after tumour cell injection. *, p< NATURE MATERIALS

19 SUPPLEMENTARY INFORMATION Fig. S12. Cells from 3D soft fibrin gels but not from rigid plastic express Sox2. (a) 10 5 B16- F1 cells cultured in 3D soft fibrin gels (3D; 90-Pa) or on rigid plastic (Contr) were used for total RNA isolation. The expressions of c-myc, Rex-1, and Sox2 were analyzed by RT-PCR. The first round PCR (1 st ) product was used as the DNA template for the second round PCR (2 nd ). Note that after 2nd round PCR, Sox2 is still un-detectable in Contr but amplified in 3D, suggesting that Sox2 was only expressed in cells cultured from 3D soft fibrin gels. Similar results were observed from 3 independent experiments. (b) Self-renewal marker Sox2 of B16-F1 cells was quantified by real-time PCR. Same mrna sample of B16-F1 tumour spheroid cells was used as above. 3D B16F1: 90-Pa soft 3D fibrin gels; Control B16F1: 2D rigid plastic dishes. Mean ± s.e.m.; n=6 samples for each condition from 2 independent experiments. **p <0.0073, compared with control cells. NATURE MATERIALS 19

20 Fig. S13. Knocking down Sox2, c-kit, Nestin, or Bmi-1 in B16-F1 spheroids on 2D soft fibrin gels promotes colony spreading. B16-F1 cells were seeded onto 2D soft fibrin gels (90- Pa) in 24-well plates and cultured for 3 days. Sox2, c-kit, Nestin or Bmi-1 sirna and the corresponding scrambled control oligonucleotides (all images of minus signs) were transfected to the cells. The colonies were imaged at different time points post sirna (left, middle, and right columns are before sirna transfection, 24 hr post-transfection, and 48 hr post-transfection, respectively). It appears that sirna transfection leads to spreading of cells from the spheroids, suggesting inhibition of self-renewal and onset of differentiation of these cells. (a), The images shown were representatives in each group. (b), The circularity of colonies was calculated using the formula of 4 [area]/[perimeter] 2. A perfect circle has a value of 1.0. Mean+s.e.m.; n=20 randomly chosen colonies per condition from 3 independent experiments; diamond represents controls, square represents those treated with sirna. *, p<0.05; **, p< NATURE MATERIALS

21 SUPPLEMENTARY INFORMATION Fig. S14. Cells from 3D soft fibrin gels are more resistant to apoptosis induced by cisplatin than those from the rigid plastic. B16-F1 cells were cultured in 3D soft fibrin gels (90 Pa) or on the rigid plastic. Different concentrations of cisplatin were added to the B16-F1 cell culture medium during the last 18 hr of 5-day culture. Cells were collected and stained with FITC conjugated Annexin-V for apoptotic detection by flow cytometry. Mean ± s.e.m., n=3 independent experiments; *p <0.05, compared with control cells. NATURE MATERIALS 21

22 Fig. S15. Similar F-actin distribution of 3D fibrin gel cultured B16-F1 cells as control cancer cells. Six hours after re-plating B16-F1 cells (cultured for 5 days in soft 3D fibrin gel or on rigid dish) on the dish pre-coated with collagen-1, F-actin of the cells was stained by TRITClabeled phalloidine. Nucleus was stained with Hoechst NATURE MATERIALS

23 SUPPLEMENTARY INFORMATION Fig. S16. No differences in expressions of integrins between 3D fibrin gel-cultured and control B16-F1 cells B16-F1 cells cultured in 3D soft fibrin gels or on the rigid plastic were used for total RNA isolation. The expressions of α v, β 3, α 5 and, β 1 integrin genes were analyzed by RT-PCR. No apparent differences were observed between the two culture conditions. Similar results were obtained in 3 independent experiments. NATURE MATERIALS 23

24 Fig. S17. B16-F1 cells form and grow spheroid colonies via v 3 integrins in 3D fibrin gels. Blocking α v β 3, but not β 1 integrin, with specific antibody inhibited spheroid colony formation and growth within 90-Pa fibrin gels. (a) Colony number as a function of culture time from Day 1 to Day 5 when treated with α v β 3 or β 1 blocking antibodies. No significant differences are observed between Control and Ab-β 1 (p>0.40 for all days), or between Control and 1 µg/ml Abα v β 3 (p>0.20 for all days except Day 4 where p<0.04), or between Control and 20 µg/ml Ab-α v β 3 at Day 1 and at Day 5 (p>0.1), or between Control and 100 µg/ml Ab-α v β 3 at Day 1 (p>0.07).. There are significant differences between Control and 20 µg/ml Ab-α v β 3 at Day 2, 3, and 4 (all p<0.043), between Control and 100 µg/ml Ab-α v β 3 at Day 2 through Day 5 (all p<0.01). (b) Colony size as a function of culture time. There are no significant differences between Control and 100 µg/ml Ab-β 1 or between Control and 1 µg/ml ab-α v β 3 (all p>0.05). There are significant differences between Control and 20 (or 100) µg/ml ab-α v β 3 (all p<0.001 except at Day 1 for 20 µg/ml ab-α v β 3 ). Mean+s.e.m.; n=3 independent experiments. 24 NATURE MATERIALS

25 SUPPLEMENTARY INFORMATION Fig. S18. RhoA-mediated cell contractility is associated with round colony formation and growth within 3D fibrin gels. Formation and growth of round colonies in 3D (a, b) and on 2D fibrin gels (c, d) were inhibited by Y-27632, a ROCK inhibitor in a dose-dependent manner. In (a), there are significant differences between control and 5, or 25, or 50 µm Y from Day 1 through Day 5 (all p<0.04). In (b), there are significant differences between control and 5, or 25, or 50 µm Y at Day 1 (all p<0.023), at Day 2 (all p<0.0044), Day 3 (p< except 5 µm, where p>0.25), Day 4 (p< except 5 µm, where p>0.11), Day 5 (all p<0.0012). In (c), there are significant differences between Control and 5, or 25, or 50 µm Y (all p<0.05 except at Day 1 for 25 µm). In (d), there are significant differences between Control and 5, or 25, or 50 µm Y (all p<0.01). Mean+s.e.m.; n=3 independent experiments. NATURE MATERIALS 25

26 Fig. S19. Similar transmigration rates for soft 3D fibrin gel cultured cells and control B16- F1 cells. In vitro transwell assays were performed and cell transmigration rates were compared between the cells isolated from tumour spheroids cultured in soft 3D fibrin gels for 5 days and control B16-F1 cells. 15,000 cells per well were seeded on the upper chamber of the transwell membrane and cell numbers were counted 3, 6, or 9 hr after 5% serum (chemotactic factors) was added to the lower chamber. The membranes were pre-coated with collagen-1. There were no significant differences between 3D cells and control cells (p>0.72, 0.19, 0.34 for 3, 6, or 9 hr). Mean+s.e.m.; n=3 independent experiments. 26 NATURE MATERIALS

27 SUPPLEMENTARY INFORMATION Fig. S20. Fibrin/Fibrinogen is present in the stroma of B16-F1 melanoma. B16-F1 cells from the rigid plastic were inoculated at 10 5 per mouse subcutaneously to C57BL/6 mice. After tumour formation, tumour tissues were collected for immunohistochemical staining to detect the expression of fibrin/fibrinogen (brown color represents positive tissues for fibrin/fibrinogen). Tissues from two different mice (Melanoma #1, #2) were presented. Control: the isotype IgG. Fibrin Ab: antibodies against fibrin/fibrinogen. NATURE MATERIALS 27

28 Fig. S21. AKT is constitutively active in cells from 3D soft fibrin gels. B16-F1 cells were cultured on rigid plastic, in 3D soft fibrin gels (90 Pa), or in 3D soft collagen gels (94 Pa). On Day 5, 20 ng/ml EGF was added to the culture media for 20 min. Single cell suspensions were collected from these different culture conditions, stained with Alexa Fluor 488-conjugated antiphosphorylated AKT1/2 antibody, and analyzed by flow cytometry. (a) Representatives of FACS profiles. (b) Mean Fluorescence Intensity (MFI) from three independent experiments (Mean+s.e.m.). 28 NATURE MATERIALS

29 SUPPLEMENTARY INFORMATION Fig. S22. AKT inhibition leads to decreases in spheroid growth. B16-F1 cells were cultured in 3D soft fibrin gels (90 Pa). Two days later, cells were treated with the selective AKT1/2 inhibitor (A6730 at 1 µm or 10µM). Cells treated with PBS were used as control. The spheroids were imaged on Day 5 (all images were taken at 4x objective). (a) Representative images of the colonies. (b) The average colony projected area from 3 independent experiments (Mean+s.e.m.; n=20 randomly chosen colonies per condition; control (Con) colony areas were set to 1.0). NATURE MATERIALS 29