Organotypic 3D Invasion Assay
Materials Required
Principle
The organotypic 3D invasion assay measures carcinoma-cell invasion into a fibroblast-remodeled extracellular matrix, usually collagen I with or without basement-membrane matrix, under an air-liquid or grid-supported culture condition; the readout is invasion depth, invaded area, or an invasion index from histological or fluorescence images[1][2][3]. This assay models stromal regulation of invasion because fibroblasts or CAFs remodel matrix, generate tracks, and can lead collective carcinoma-cell invasion; the resulting cancer-cell penetration into the gel reflects tumor-stroma-ECM interactions rather than migration on a rigid 2D substrate[1][4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use DMEM-based fibroblast and cancer-cell media, FBS, PBS, trypsin-EDTA, collagen I, Matrigel or another Engelbreth-Holm-Swarm basement-membrane matrix when following the breast-cancer organotypic version, PFA/glutaraldehyde fixative, ethanol, paraffin-embedding reagents, and H&E reagents for histological invasion analysis[1].
• For collagen-I-only organotypic gels, use rat-tail collagen I, MEM or DMEM-based medium, NaOH for neutralization, FCS/FBS, PBS, trypsin, and 4% PFA fixation; collagen gels are contracted by embedded fibroblasts before tumor cells are seeded on top[2].
• H&E staining is used to visualize tumor-cell position relative to the matrix surface, and immunohistochemistry, immunofluorescence, or fluorescently labeled tumor/stromal cells may be used when cell identity or live-cell visualization is required[1][2].
• Required equipment includes tissue-culture plates, sterile forceps/spatulas/scalpels, nylon filters when using the breast-cancer protocol, stainless-steel grids or bridges to support gels at the medium interface, a CO2 incubator, histology-processing equipment, a bright-field microscope with 10× or 20× objective, and ImageJ or equivalent image-analysis software[1][2].
• For collagen-I-only organotypic gels, use rat-tail collagen I, MEM or DMEM-based medium, NaOH for neutralization, FCS/FBS, PBS, trypsin, and 4% PFA fixation; collagen gels are contracted by embedded fibroblasts before tumor cells are seeded on top[2].
• H&E staining is used to visualize tumor-cell position relative to the matrix surface, and immunohistochemistry, immunofluorescence, or fluorescently labeled tumor/stromal cells may be used when cell identity or live-cell visualization is required[1][2].
• Required equipment includes tissue-culture plates, sterile forceps/spatulas/scalpels, nylon filters when using the breast-cancer protocol, stainless-steel grids or bridges to support gels at the medium interface, a CO2 incubator, histology-processing equipment, a bright-field microscope with 10× or 20× objective, and ImageJ or equivalent image-analysis software[1][2].
Experimental Procedure
• Prepare fibroblasts or CAFs as single-cell suspensions and embed them in the matrix; in the breast-cancer protocol, a 1 mL gel contains FBS, 5× DMEM, fibroblast culture medium, Matrigel, collagen I, and fibroblasts, with final collagen I and Matrigel concentrations of 4 mg/mL and 2 mg/mL, respectively[1].
• For the collagen-I-only protocol, mix cold collagen I with medium components and neutralize to approximately pH 7.2, add fibroblasts, dispense about 2.5 mL per dish, set at 37 °C for 10 min, add fibroblast medium, detach the gel from the dish edge, and allow fibroblast-mediated contraction for about 6-8 days until the gel diameter decreases from about 3.5 cm to 1.5 cm[2].
• Prepare carcinoma cells as a single-cell suspension; the breast-cancer protocol reports 5 × 106 cells/mL and seeding 100 μL on top of each gel, followed by 6-8 h adhesion at 37 °C and 5% CO2[1].
• For the breast-cancer organotypic assay, keep gel components on ice, cast 900 μL gel per 24-well plate well, set the gel for 1 h at 37 °C and 5% CO2, add 1 mL medium, incubate overnight, seed cancer cells on top, allow adhesion, transfer the gel onto a gel-coated nylon filter supported by a metal bridge, add medium until it contacts the filter from below, overlay the cancer-cell layer with 100 μL cell-free gel, culture for 5 days at 37 °C and 5% CO2, and change medium daily[1].
• For the collagen-I-only organotypic assay, transfer the contracted fibroblast-collagen matrix to a 24-well dish, seed approximately 4 × 104 tumor cells/mL in 1 mL medium on top, allow tumor cells to reach confluence over about 3-5 days, place the matrix on a sterile grid at an air-liquid interface, maintain medium contact with the underside of the gel without submerging it, replace medium every 2 days, and analyze invasion from day 1 to day 21 or longer depending on the biological question[2].
• Terminate the assay by fixation; the breast-cancer protocol fixes gels overnight at 4 °C in 4% PFA plus 1% glutaraldehyde, washes three times for 10 min in PBS, halves the gel, stores one half in 70% ethanol, and processes the other half for paraffin embedding and H&E staining[1].
• Acquire 5-7 central images per gel using bright-field microscopy at 10× or 20× magnification, avoiding gel edges because fibroblasts can concentrate at borders and create invasion artifacts[1].
• Quantify invasion by measuring non-invaded cancer-cell area and total cancer-cell area in ImageJ or equivalent software; invaded area is calculated from these regions, and the invasion index is reported as invaded area divided by total cancer-cell area[1][3].
• Use fibroblast-free gels as a negative matrix-remodeling control, normal fibroblast-containing gels as a stromal comparator, CAF-containing gels as a pro-invasive stromal condition, and the “killing assay” variant when testing whether fibroblast-remodeled tracks alone can support subsequent cancer-cell invasion[1][4][5][6].
• For the collagen-I-only protocol, mix cold collagen I with medium components and neutralize to approximately pH 7.2, add fibroblasts, dispense about 2.5 mL per dish, set at 37 °C for 10 min, add fibroblast medium, detach the gel from the dish edge, and allow fibroblast-mediated contraction for about 6-8 days until the gel diameter decreases from about 3.5 cm to 1.5 cm[2].
• Prepare carcinoma cells as a single-cell suspension; the breast-cancer protocol reports 5 × 106 cells/mL and seeding 100 μL on top of each gel, followed by 6-8 h adhesion at 37 °C and 5% CO2[1].
• For the breast-cancer organotypic assay, keep gel components on ice, cast 900 μL gel per 24-well plate well, set the gel for 1 h at 37 °C and 5% CO2, add 1 mL medium, incubate overnight, seed cancer cells on top, allow adhesion, transfer the gel onto a gel-coated nylon filter supported by a metal bridge, add medium until it contacts the filter from below, overlay the cancer-cell layer with 100 μL cell-free gel, culture for 5 days at 37 °C and 5% CO2, and change medium daily[1].
• For the collagen-I-only organotypic assay, transfer the contracted fibroblast-collagen matrix to a 24-well dish, seed approximately 4 × 104 tumor cells/mL in 1 mL medium on top, allow tumor cells to reach confluence over about 3-5 days, place the matrix on a sterile grid at an air-liquid interface, maintain medium contact with the underside of the gel without submerging it, replace medium every 2 days, and analyze invasion from day 1 to day 21 or longer depending on the biological question[2].
• Terminate the assay by fixation; the breast-cancer protocol fixes gels overnight at 4 °C in 4% PFA plus 1% glutaraldehyde, washes three times for 10 min in PBS, halves the gel, stores one half in 70% ethanol, and processes the other half for paraffin embedding and H&E staining[1].
• Acquire 5-7 central images per gel using bright-field microscopy at 10× or 20× magnification, avoiding gel edges because fibroblasts can concentrate at borders and create invasion artifacts[1].
• Quantify invasion by measuring non-invaded cancer-cell area and total cancer-cell area in ImageJ or equivalent software; invaded area is calculated from these regions, and the invasion index is reported as invaded area divided by total cancer-cell area[1][3].
• Use fibroblast-free gels as a negative matrix-remodeling control, normal fibroblast-containing gels as a stromal comparator, CAF-containing gels as a pro-invasive stromal condition, and the “killing assay” variant when testing whether fibroblast-remodeled tracks alone can support subsequent cancer-cell invasion[1][4][5][6].
Troubleshooting
Problem: Gel damage during transfer.
• Possible Cause: Soft gels can be damaged by direct contact with metal bridges.• Literature-supported Solution: Place a gel-coated nylon filter between the organotypic gel and the metal bridge before culture at the medium interface[1].
Problem: Variable invasion near gel borders.
• Possible Cause: Fibroblasts can accumulate at gel edges and produce local artifacts.• Literature-supported Solution: Capture images from the central region of each gel for quantification[1].
Problem: Poor or inconsistent gel contraction.
• Possible Cause: Collagen concentration, fibroblast density, fibroblast culture, and collagen batch affect contraction rate.• Literature-supported Solution: Optimize collagen concentration, fibroblast number, and contraction time for the specific fibroblast source and matrix batch[1][2].
Problem: Bubbles or inconsistent gel quality.
• Possible Cause: Collagen/Matrigel mixtures are viscous and can trap bubbles or remain in pipette tips.• Literature-supported Solution: Keep matrix components cold, pipette carefully, avoid bubble formation, and prepare excess gel mixture to reduce pipetting error[1].
References:
- [1]. Ranftl RE, et al. Analysis of breast cancer cell invasion using an organotypic culture system. Methods Mol Biol. 2017;1612:199-212. [Content Brief]
- [2]. Timpson P, et al. Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context. J Vis Exp. 2011;(56):e3089. [Content Brief]
- [3]. Nyström ML, et al. Development of a quantitative method to analyse tumour cell invasion in organotypic culture. J Pathol. 2005;205(4):468-475. [Content Brief]
- [4]. Gaggioli C, et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 2007;9(12):1392-1400. [Content Brief]
- [5]. Calvo F, Ege N, Grande-Garcia A, Hooper S, Jenkins RP, Chaudhry SI, et al. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat Cell Biol. 2013;15(6):637-646. [Content Brief]
- [6]. Scott RW, Hooper S, Crighton D, Li A, König I, Munro J, et al. LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells. J Cell Biol. 2010;191(1):169-185. [Content Brief]
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