Engineering tumors with 3D scaffolds

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1 Engineering tumors with 3D scaffolds Claudia Fischbach, Ruth Chen, Takuya Matsumoto, Tobias Schmelzle, Joan S Brugge, Peter J Polverini & David J Mooney Supplementary figures and text: Supplementary Figure 1 Tissue formation in 3D PLG scaffolds Supplementary Figure 2 ECM expression in 2D and 3D PLG scaffolds Supplementary Figure 3 Angiogenic characteristics Supplementary Figure 4 Spheroid formation in 3D Matrigel culture Supplementary Figure 5 Flow cytometry of integrins Supplementary Figure 6 N-cadherin expression Supplementary Figure 7 E-cadherin expression Supplementary Methods

2 Supplementary Figure 1: Tissue formation in PLG scaffolds. Confocal analysis of tumor cells cultured within PLG scaffolds. Staining with calcein green and propidium iodide using the CARV II image acquisition system (BD Biosciences) and IPLab image analysis software (Scanalytics) demonstrated formation of tissues composed of living (green fluorescence) and dying (red fluorescence) cells, respectively. The image represents a 5 um z-stack at the top of a whole cell polymer construct at an early time point of PLG scaffold culture (day 5), and, hence, a relatively small number of dead cells was detected. A photomicrograph of a representative H&E stained histological cross-section is shown at a similar scale to correlate the localization of viable and dead areas within PLG scaffold cultures at this time-point. Scale bars represent 5 um.

3 Supplementary Figure 2: ECM expression in and PLG scaffolds. Laminin 4 days 9 days 4 days 9 days Fibronectin 4 days 9 days 4 days 9 days Western Blot analysis demonstrated that laminin deposition within PLG scaffold cultures is reduced relative to monolayer culture, while fibronectin deposition is increased.

4 Supplementary Figure 3: Angiogenic characteristics a) VEGF (pg/1, cells/24h) VEGF bfgf IL-8 3 % O2 () 2% O2 () bfgf (pg/1, cells/24h) % O2 () 2% O2 () IL-8 (pg/1, cells/24h) % O2 () 2% O2 () b) Protein secretion (pg/1, cells/24h) VEGF IL-8, with Matrigel, without Matrigel c) MCF-7 Relative increase * d) f (protein day 1) In vivo VEGF IL-8 VEGF IL-8 1 day 5 days 1 days 15 days 2 days U87 Relative increase VEGF IL-8 f (protein day 5) f (protein day 5) days 1 days 15 days 5 days 1 days 15 days e) control EGM-2 Max. sprout length (um) control EGM-2 (a) OSCC-3 were cultured in typical monolayer at normoxia, in PLG scaffolds at normoxia, and in monolayer at or 2% O2 levels to ascertain the effects of PLG scaffold culture and hypoxia. Cells significantly increased secretion of VEGF, bfgf, and IL-8 in response to PLG scaffold culture. Hypoxia also significantly elevated VEGF and bfgf secretion, but only modestly upregulated IL-8. (continued on next page)

5 Suppl Fig. 3 continued. (b) ELISA analysis of conditioned media collected from culture (2- D), PLG scaffold culture with inclusion of Matrigel during cell seeding (, with Matrigel) or without addition of Matrigel (, without Matrigel) revealed that VEGF and IL-8 secretion were up-regulated under both PLG scaffold conditions, as compared to, and that both PLG scaffold conditions enhanced IL-8 secretion more significantly, relative to VEGF. Data represent mean ± SD (n=3). Data and error bars are small where not visible. P <.1. (c) PLG scaffold culture of MCF-7 breast cancer cells and U87 glioblastoma cells resulted in upregulation of VEGF and IL-8 relative to culture, and IL-8 levels were modulated more significantly than VEGF. Data represent mean ± SD (n=4). Data and error bars are small where not visible. * P <.5, P <.1. (d) The cell-associated VEGF and IL-8 in OSCC-3 were quantified as a function of time (normalized to day 1 [in vivo] and day 5 [in vitro] levels), and IL-8 was up-regulated more significantly in PLG scaffolds and in real tumors in vivo than in culture. Tumor cells situated within PLG scaffolds in vitro and in vivo secreted VEGF and IL- 8 in a similar manner, whereas in monolayer culture the in vivo temporal profile was mimicked only slightly. Error bars are small where not visible. (e) The ability of HUVECs to initiate angiogenesis in vitro, as measured with a typical sprouting assay, was significantly enhanced by exposure to either a positive control (EGM-2 media containing high concentrations of growth factors) or to conditioned media from PLG scaffold culture. A control condition (basal media) and conditioned media from standard monolayer culture () was also tested. The length of the sprouts was greatest when exposed to conditioned medium from PLG scaffold cultures. Scale bars represent 1 um. All data represent mean ± SD (n=4). *P <.5, P <.1.

6 Supplementary Figure 4 Spheroid formation in Matrigel culture. Day 1 Day 4 Day 7 Day 1 # of spheroids < ±2.44 um Diameter (um) >16 Matrigel culture of initially dispersed OSCC-3 cells (day 1) resulted in formation and growth of multicellular tumor spheroids over time (day 4, 7, 1) that exhibited a size distribution of 99 ±2 um at day 1 after seeding as detected by image analysis. Scale bars represent 1 um.

7 Supplementary Figure 5 Flow cytometry of integrins. a) alpha 5 beta 1 % of Max % of Max b) alpha 6 beta 4 % of Max % of Max antibody control antibody control (a) FACS analysis of fibronectin receptor (alpha5beta1 integrin) expression revealed that subunit alpha5 was significantly up-regulated by PLG scaffold culture, while beta1 expression was altered only marginally by PLG scaffold culture. (b) FACS analysis of laminin receptor (alpha6beta4 integrin) expression demonstrated that expression of alpha6 was not altered by PLG scaffold culture, while subunit beta4 was marginally up-regulated. An antibody control ( and antibody control) is also shown confirming specificity of the antibody staining.

8 Supplementary Figure 6 N-cadherin expression. (Propidium Iodide) control.8% (FITC) (Propidium Iodide) 1.6% (FITC) (Propidium Iodide) 2.8% (FITC) PLG scaffold culture led to up-regulation of N-cadherin as determined by flow cytometry. Tumor cell populations cultured in conventional culture () and PLG scaffold culture (3- D) contained 1.6% and 2.8% of N-cadherin-positive cells, respectively. An antibody control condition (control) was also measured and confirmed specificity of immunostaining. For analysis, fluorescence gates (R2) were set to include cells that were both propidium iodide negative (i.e., live cells) and FITC-positive (i.e., N-cadherin expressing cells).

9 Supplementary Figure 7 E-cadherin expression. OSCC-3 MCF-7 5 um Immunocytochemistry for E-cadherin showed that OSCC-3 lack expression of E-cadherin. Specificity of staining was confirmed by cytochemical analysis of E-cadherin positive MCF-7 breast cancer cells.

10 Supplementary Methods Immunofluorescence and Western Blotting. tumor structures cultured on Matrigel were analyzed via immunofluorescense as previously described 1. Briefly, structures were fixed in 2% paraformaldehyde and incubated at 4 C overnight with primary antibodies raised against Ki67 (Zymed), beta-catenin (BD Biosciences), laminin V (Chemicon), and active caspase 3 (Cell signaling). Incubation with Alexafluor conjugated secondary antibodies (Molecular Probes) was performed for 1h at RT and followed by incubation with Topro-3 (Molecular Probes) and DAPI (Sigma) before mounting with the Prolong Antifade Reagent (Molecular Probes). Confocal analysis was performed using a Nikon E8 with Bio-Rad confocal imaging system (Nikon Imaging Center, Harvard Medical School). For Western analysis, cells and constructs (seeded without Matrigel) were lysed in Ripa buffer containing protease inhibitors. Protein concentrations were determined with a BCA kit (Pierce) and equal amounts of protein were loaded in gels (Biorad), resolved by SDS-PAGE, and transferred to PVDF membranes (Biorad). Membranes were probed at RT for 1h with polyclonal, non-isoform specific anti-human primary antibodies (laminin: Novus Biologicals, fibronectin: Sigma) and 45 min with a species-specific HRP-conjugated secondary antibody (Novus Biologicals), followed by ECL detection (Amersham Biosciences). Densitometric analysis of developed films was performed with ImageJ software and values were normalized to adsorbed serum proteins. Analysis of cell adhesion molecules. For flow cytometry of integrin subunits and N-cadherin cells were enzymatically harvested using collagenase/dispase, blocked for nonspecific binding (6 min PBS,.5% BSA), incubated with 1 ug anti-human alpha5, beta1, beta4, N-cadherin antibody/1 6 cells (all from BD Biosciences) for 1h at 4ºC, washed 3 times, and labeled with AlexaFluor 488-conjugated goat immunoglobulin (Molecular Probes) for 45 min. Controls were IgG isotype controls (BD Biosciences). Staining for alpha6 was conducted with a FITCconjugated rat anti-human primary antibody (BD Biosciences). After washing three times in PBS/.5% BSA and counterstaining with 1 ug/ml propidium iodide, cells were analyzed on a BD LSRII and data processed using FlowJo 7..6 software (Tree Star Inc.). Immunocytochemistry for E-cadherin was performed using a mouse anti E-cadherin IgG (BD), a secondary goat anti mouse HRP labeled antibody (Dako), and DAB detection (Biocare Medical). ELISAs and conditioned media experiments. and cultures were washed twice with PBS followed by incubation with DMEM/1% FBS. After 24 h media were collected and VEGF, bfgf, IL-8 ELISAs (R&D) were performed according to manufacturer s instructions; values were normalized to cell numbers as determined by Hoechst33258 DNA assay. Protein from in vivo tumors was extracted by tissue homogenization with TPER lysis buffer (Pierce), measured by ELISA, and standardized for total protein content as determined via Biorad protein assay. For conditioned media experiments, media were collected, normalized to the same cell number, and concentrated 1-fold using Amicon Ultrafree 15 (Millipore); volume was then brought back to 5-fold concentration by diluting with EBM (Cambrex) containing 2% FBS to result in VEGF concentrations in (both PLG and Matrigel) media of 2 ng/ml. Neutralizing VEGF antibody (R&D) was added at.4 ug/ml, a concentration determined to completely inhibit proproliferative effects of 2 ng/ml VEGF (see Supplementary Fig. 8 online), while neutralizing IL-8 antibody (R&D) was added at a concentration of 1 ug/ml. HUVEC (Cambrex) proliferation was investigated in 12-well plates by cell counting after 4 days. Tube formation was assessed in chamber slides coated with growth factor reduced Matrigel (BD Biosciences) as previously described 23.

11 Histological analysis. For histological investigations, engineered tumor constructs and explants were placed in 4% paraformaldehyde overnight, and stored in 7% ethanol before sectioning for histology. Hematoxylin&eosin (H&E) staining was routinely performed. To identify blood vessels, tissue sections were subjected to proteinase K (Dako) antigen retrieval and subsequently immuno-stained with antibodies raised against mouse CD31 (Pharmingen) using the TSA Biotin System (Perkin Elmer) and anti-rat secondary antibody (Vector Laboratories). The number and perimeter of blood vessels with lumen were quantified by capturing digital images of tissue sections, analyzed with ImageJ software, and normalized to tissue area. Following enzymatic antigen retrieval alpha-sma staining was performed with a primary anti-human antibody (Abcam) and the EnVision+ System-HRP (DAB) kit (Dako). Statistical analysis. ANOVA with Tukey postanalysis was used to test for statistical significance between experimental groups. Four independent experiments were performed for each condition tested in vitro, unless otherwise stated. Statistical significance of in vivo samples was tested with n=8 samples. In all figures, error bars indicate standard deviations (*, P <.5;, P <.1). Reference 1. Debnath,J., Muthuswamy,S.K. & Brugge,J.S. Morphogenesis and oncogenesis of MCF- 1A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 3, (23).