HSC-3 cells mono- and Co-cultures using the 3D organotypic myoma model
This study was approved by the Ethics Committee of the Oulu University Hospital, Oulu, Finland.
The 3D organotypic myoma model was based on uterine leiomyoma tissue that was obtained from routine surgical operations after informed consent of the donors. The model was previously described in detail [
19]. Homogeneous areas of the myoma served as sources for preparing discs for the organotypic culture by first cutting 3-mm slices with a disposable scalpel and further into discs with an 8-mm biopsy punch (Kai Industries Co., Gifu, Japan). The myoma discs were stored at −70°C in media with 10% DMSO (Sigma Aldrich). For preparing myoma-based organotypic cultures, myoma discs were equilibrated in media at room temperature (RT) for 1 hour. The myoma discs were placed into Transwell inserts (diameter, 6.5 mm; Corning, Inc., Corning, NY). For preparing mono-cultures, 3–4 × 10
5 cells (e.g., oral tongue SCC HSC-3 cells, Japanese Collection of Research Bioresources Cell Bank, Japan) were cultured on the top of myoma tissue and allowed to attach overnight, and the myoma discs were removed from the Transwell inserts and transferred onto uncoated nylon discs resting on curved steel grids (3 × 12 × 15 mm) in 12-well plates with sufficient volume of media (1 ml). The myoma organotypic cultures were maintained for 14 days, and the media were changed according to the collagen organotypic culture protocol. In the co-culture assays, 2–3 × 10
5 cancer cells were first mixed with 4 × 10
5 other cell types (e.g., fibroblasts) and then cultured on myoma tissue and further processed as the mono-cultures [
3,
19].
In addition, rinsed myoma (RM) discs were prepared by rinsing discs in DMEM (Sigma Aldrich, Ayrshire, UK), gently rotating them at 4°C for two weeks and changing the media every three days [
20]. The culturing procedures on the RM discs were identical to the above-described methodology.
Experiments were performed in normoxic and two types of hypoxic conditions: (a) 1% O
2 hypoxia created by exposure of the assays in a hypoxic chamber (InVivo2 400, Ruskinn Technology, UK) for 20 h, and (b) overnight exposure to 100 mM cobalt chloride (CoCl
2), a known hypoxia-mimicker, according to Brusewold et al. [
21] and detailed elsewhere [
20]. More specifically, HSC-3 monocultures were performed on both intact myoma (IM) and RM discs in both normoxic and hypoxic conditions. Co-cultures were performed on IM discs in normoxia.
HSC-3 organotypic mono-cultures and immunohistochemical stains
The RM discs that had been mono-cultured with HSC-3 (3–4 × 10
5 cells) at 37°C with 5% CO
2 for 14 days [
3,
20] were studied for the immunohistochemical expression of CAV1 and αSMA (serial sections). The same mono-cultures were also performed on the IM followed by the same immunostaining. RM/IM discs were fixed in 4% neutral-buffered formalin and embedded in paraffin [
3,
20]. Five micron-thick sections were immunostained with anti-caveolin-1 (E294, rabbit monoclonal, Abcam, Cambridge, UK, 1:250) or with anti-αSMA (1A4, mouse monoclonal, Dako, Glostrup, Denmark, 1:1000). Staining was demonstrated by a Dako REAL EnVision kit (peroxidase/DAB+, Dako) for mouse and rabbit primary antibodies. The above-described procedures on RM and IM HSC-3 mono-cultures were performed in both normoxic and hypoxic (1%O
2) conditions [
20].
IM and RM HSC-3 mono-cultures in normoxic and hypoxic conditions were triple immunostained with CAV1 antibody (1:100), pan-cytokeratin (1:50) and Twist (1:250; ab50581, Abcam, Cambridge, UK). A mixture of equal volumes of CAV1 and cytokeratin antibodies was incubated overnight at 4°C, rinsed and added to double-stain polymer adjusted for both mouse (HRP) and rabbit (AP) antibodies (Innovex Biosciences, Pinoles, CA, USA) for 30 min at RT. Sections were then stained with DAB for visualizing cytokeratin colored brown and with fast red (Permanent AP red kit, Zytomed, Berlin, Germany) for visualizing CAV1 colored purple. Antigen retrieval for Twist was performed by heating the slides in sterile water at 92° for 10 min and cooling for 20 min, followed by exposure to the Twist antibody at 4°C overnight and thereafter conjugation with rabbit/mouse polymer (Super-picture HRP, Invitrogen, Frederick, MD, USA) for 30 min at RT. Twist was finally visualized colored green (Permanent HRP green kit, Zytomed, Berlin, Germany).
The other series of triple immunostaining comprised cytokeratin (colored brown), Twist (colored purple) and alpha-smooth muscle actin (αSMA, colored green; 1:50; 1A4, Dako, Glostrup, Denmark). The procedure started with the mixing of equal volumes of Twist and cytokeratin antibodies followed by αSMA.
Assessment of the triple-immunostained sections was only qualitative and intended to demonstrate the localization of the staining among the cell types. Since the triple immunostaining revealed a similar pattern of expression of CAV1 in both RM and IM assays in normoxic and hypoxic conditions, the co-culture studies were performed only on the IM discs in normoxic conditions.