Overview of methodology, tools and reagents for evaluating cell proliferation and invasion using multicellular tumor spheroids.

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1 The Next Step in the Evolution of 3D Culture: Utilizing Extracellular Matrix to Enhance Multicellular Tumor Spheroid Models for Proliferation and Invasion Overview of methodology, tools and reagents for evaluating cell proliferation and invasion using multicellular tumor spheroids.

2 TOOLS AND REAGENTS

3 3-D SPHEROID PROLIFERATION/VIABILITY

4 Sample Preparation for Spheroid Formation Calculate volumes for 10X Spheroid Formation ECM, Complete Growth Medium, and Cells (1 x 10^6 cells/ml): Reagent 1 Well 3,000 Cells/Well 10X Spheroid Formation ECM (4 C) 5 µl 5 µl Cells (at 1 x 10^6 cells/ml) X µl 3 µl Complete Growth Medium (4 C) 45-X µl 42 µl Total 50 µl 50 µl

5 Sample Preparation for Spheroid Formation 10X Spheroid Formation ECM Complete Growth Medium Thaw 10X Spheroid Formation ECM on ice at 4 C overnight. Divide into working aliquots; store at -80 C for optimal stability, avoid freeze-thaws. Keep on ice throughout procedure. Dilute with cold (4 C) complete growth medium, and pipet up and down to mix. Spheroid Formation ECM

6 Sample Preparation for Spheroid Formation Harvest Centrifuge Cells at 80% Confluence Spheroid Formation ECM Resuspend Cells at 1 x 10^6 cells/ml Culture cells to 80% confluence. Harvest cells, centrifuge, and aspirate medium. Resuspend at 1 x 10^6 cells/ml in complete growth medium. Add the calculated amount of cells to the tube containing Spheroid Formation ECM. Cells in Spheroid Formation ECM

7 Sample Preparation for Spheroid Formation Cells in Spheroid Formation ECM 96 Well Spheroid Formation Plate Add 50 µl of the cells in Spheroid ECM to the 96 Well Spheroid Formation Plate. Centrifuge at 200 x g for 3 minutes in a swinging bucket rotor. Incubate plate with cells at 37 C, 5% CO 2 for 72 hours. Cells and Spheroid Formation ECM in 96 Well Spheroid Formation Plate

8 Sample Preparation for Spheroid Formation Spheroid formed within 72 hours Add 50 µl of 2X treatment. Cells assemble into one spheroid/well within 72 hours. Prepare solutions containing 2X treatment compounds in complete growth medium. Add 2X treatment (50 µl) to each well. Incubate at 37 C, 5% CO2 for desired treatment period. Incubate for desired treatment period.

9 Image Analysis Evaluation of Spheroid Growth Image Analysis

10 3-D Culture Spheroid Proliferation/Viability MDA-MB D Spheroid Growth A B Area (µm 2 ) Days MDA-MB-231 Breast cancer cells in spheroid culture for 3 days form A) loose colonies in media and B) tight colonies in media + ECM. 3-D spheroid cultures are large cell aggregates which exhibit physiological characteristics of small tumors. Incorporation of ECM proteins is necessary to promote in vivo cell behavior. This assay provides an in vitro model for evaluating tumor growth in response to pharmacological agents.

11 3-D Culture Spheroid Proliferation/Viability Image Analysis % Area (µm 2 ) y = 23918x R² = Area (% untreated) 100% 80% 60% 40% y = ln(x) R² = % Day % Bleomycin (µm) Spheroid growth of MDA-MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37 C, 5% CO 2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37 C, 5% CO 2. Spheroids were photographed every 24 hours, and images were analyzed using ImageJ software. Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 C, 5% CO 2 to induce spheroid formation. Spheroids were then treated with the corresponding doses of Bleomycin and incubated at 37 C, 5% CO 2 for 96 hours. Spheroids were photographed after 96 hours, and images were analyzed using ImageJ software

12 3-D Culture Spheroid Proliferation/Viability Analysis Fluorometric or Colorimetric Quantitation

13 Fluorometric Quantitation with Resazurin

14 3-D Culture Spheroid Proliferation/Viability Fluorometric Quantitation y = ln(x) R² = % 100% % RFU Viability 60% 40% y = ln(x) R² = % Spheroid Seeding Density (Cells/Well) 0% Bleomycin (um) Spheroid cell number corresponds to fluorescence output for MDA- MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37 C, 5% CO 2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37 C, 5% CO 2 for 96 hours and analyzed using Resazurin. Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 C, 5% CO 2 to induce spheroid formation. Spheroids were then treated with the corresponding doses of Bleomycin, incubated at 37 C, 5% CO 2 for 96 hours, and analyzed using Resazurin.

15 Colorimetric Quantitation with MTT

16 3-D Culture Spheroid Proliferation/Viability Colorimetric Quantitation Absorbance (570 nm) y = -1E-09x 2 + 4E-05x R² = Viability 90% 80% 70% 60% 50% 40% 30% 20% 10% y = ln(x) R² = ,000 4,000 6,000 8,000 10,000 12,000 14,000 Spheroid Seeding Density (Cells/Well) 0% -10% Bleomycin (µm) Spheroid cell number corresponds to absorbance for MDA-MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37 C, 5% CO 2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37 C, 5% CO 2 for 96 hours and analyzed using MTT. Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 C, 5% CO 2 to induce spheroid formation. Spheroids were then treated with the corresponding doses of Bleomycin, incubated at 37 C, 5% CO 2 for 96 hours, and analyzed using MTT.

17 3-D SPHEROID INVASION

18 Sample Preparation for Spheroid Invasion Invasion Matrix Thaw on ice at 4 C overnight. Spheroid in Spheroid Formation Plate Chill on ice at 4 C for 5 minutes. Cells assemble into one spheroid/well within 72 hours. Chill 96 Well Spheroid Formation Plate on ice at 4 C for 5 minutes. Working on ice, add 50 µl of Invasion Matrix to each well. Centrifuge plate at 300 x g for 5 minutes at 4 C (pre-chilled) in a swinging bucket rotor. Add Invasion Matrix and centrifuge 300 x g for 5 min at 4 C.

19 Sample Preparation for Spheroid Invasion Place Spheroid Formation Plate at 37 C, 5% CO 2. Add 100 µl of 2X treatment (37 C). Incubate for desired treatment period. Transfer the Spheroid Formation Plate to 37 C, 5% CO 2 for one hour to polymerize Invasion Matrix. Add 100 µl of complete medium with 2X invasion modulating agents (37 C) to each well. Incubate at 37 C, 5% CO 2 for desired invasion period. Analyze plate.

20 3-D Culture Spheroid Invasion DESCRIPTION Resuspend cells in spheroid formation ECM and add to round bottom wells. TIME 1-2 hours Cells assemble into compact spheroids. 3 days Add Invasion Matrix and medium containing chemoattractants and/or invasion modulating compounds. 1-2 hours Cell invade into the surrounding matrix in Response to chemoattractants 2-4 days Total Time: 5-7 days

21 3-D Culture Spheroid Invasion Analysis Bright field RFP PKH67 PKH26

22 3-D Culture Spheroid Invasion Analysis Morphology of 3D cell invasion over a four day period. Cells were seeded at 3,000 cells/well in the presence of Spheroid Formation ECM, and incubated at 37 C, 5% CO 2 for 72 hours to form spheroids. Spheroids were then embedded in 50 µl Invasion Matrix, and incubated at 37 C, 5% CO 2 for one hour for polymerization. Then 100 µl of DMEM, 10% FBS was added to each well as a chemoattractant. Non-invasive cells (MCF-7) remain as cell aggregates and do not invade into the surrounding invasion matrix; whereas, invasive cells (MDA-MB-231) invade into the surrounding invasion matrix as spindle-like protrusions.

23 3-D Culture Spheroid Invasion Analysis Quantitative analysis of surface area for non-invasive (MCF-7) and invasive (MDA-MB-231) cell lines over a four day period.

24 Different cell lines exhibit different invasive potentials and morphologies Days Post Imbedding Cell Line PC-3 MDA-MB-231 HT-1080 U-87-MG Invasion of cancer cells out of MCTS and into the surrounding invasion matrix in a time-dependent manner. Cancer cells invade into an invasion matrix composed of 10 mg/ml BME and 1 mg/ml collagen I in response to cell culture medium containing 10% FBS; wells were photographed every 24 hours over a 96 hour period.

25 Area (µm 2 ) 3-D Culture Spheroid Invasion Analysis MDA-MB D Invasion Day No Matrix Sulforaphane (µm) D culture spheroid invasion may be used to evaluate modulators of metastasis and EMT No Matrix MDA-MB-231, human breast cancer cells, were induced to form spheroids (72 hours) and embedded in Invasion Matrix. Serial dilutions of Sulforaphane were added to the DMEM, 10% FBS invasion modulating solution as indicated, and spheroid invasion was conducted over a 96 hour period. Sulforaphane inhibited MDA-MB-231 spheroid Invasion in a dose-dependent manner.

26 Summary 3-D cultures provide suitable environments for cells to selfassemble into organotypic structures mimicking the morphology and function their tissue of origin. Spheroids provide ideal tumor models based on morphology, size, physiological gradients, and the formation of heterogeneous cell populations. Spheroids exhibit an extracellular matrix which is instrumental in self-assembly of cancer cells. The addition of ECM proteins promotes spheroid formation and growth. ECM hydrogels provide physiological scaffolds for cancer cells to invade out of tumor spheroids, mimicking early events in metastasis.

27 Tools for 3-D Culture Spheroid Proliferation/Viability and Invasion Description Size Catalog # Cultrex 96 Well 3-D Spheroid BME Cell Invasion Assay 96 samples K Cultrex 96 Well 3-D Spheroid Fluorometric Proliferation/Viability Assay 96 samples K Cultrex 96 Well 3-D Spheroid Colorimetric Proliferation/Viability Assay 96 samples K

28 References 1. Harrison, R.G., ON THE STEREOTROPISM OF EMBRYONIC CELLS. Science, (870): p Scherer, W.F., J.T. Syverton, and G.O. Gey, Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med, (5): p Sutherland, R.M., et al., A multi-component radiation survival curve using an in vitro tumour model. Int J Radiat Biol Relat Stud Phys Chem Med, (5): p Kleinman, H.K., et al., Basement membrane complexes with biological activity. Biochemistry, (2): p Kawata, M., et al., Neural Rosette Formation within in Vitro Spheroids of a Clonal Human Teratocarcinoma Cell Line, PA-1/NR: Role of Extracellular Matrix Components in the Morphogenesis. Cancer Research, (10): p Benton, G., E. Crooke, and J. George, Laminin-1 induces E-cadherin expression in 3-dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status. FASEB J., (11): p Benton, G., et al., Advancing science and technology via 3D culture on basement membrane matrix. Journal of Cellular Physiology, (1): p Benton, G., et al., Multiple uses of basement membrane-like matrix (BME/Matrigel) in vitro and in vivo with cancer cells. Int J Cancer, (8): p Ivascu, A. and M. Kubbies, Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis. Journal of Biomolecular Screening, (8): p Hirschberg, H., et al., Effects of ALA-mediated photodynamic therapy on the invasiveness of human glioma cells. Lasers in Surgery and Medicine, (10): p Vinci, M.M., Advances in establishment and analysis of 3D tumour spheroid-based functional assays for target validation and drug evaluation. BMC Biology, (1): p Friedrich, J., et al., Spheroid-based drug screen: considerations and practical approach. Nat Protoc, (3): p Hirschhaeuser, F., et al., Multicellular tumor spheroids: An underestimated tool is catching up again. Journal of Biotechnology, (1): p

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