[5, 6]. Several reports support a cooperative model for cancer
invasion where distinct cell populations interact, resulting in
enhanced invasiveness and survival of the moving cell cluster [5–
7]. Cancer Associated Fibroblasts (CAFs) are one of the most
abundant cell types in solid tumors, and they play an active role in
promoting tumor invasion and metastasis. Recent studies reported
that CAFs drive the initiation of collective dissemination of epithelial tumors through ECM remodeling and intercellular interactions
mediated by adherent junction proteins [8, 9]. These findings
highlight that the stromal contribution to metastasis is not limited
to chemical crosstalk but can also be mediated by direct mechanical
interactions [9, 10].
Here we provide a three-dimensional spheroid invasion assay to
assess the invasive pattern of cocultured CAFs and cancer cells in
organotypic ECM. This method enables multicellular coculture
and is compatible with time-lapse fluorescent imaging using
spinning disc microscopy. Collective invasion patterns can be monitored over a period of 24–72 h and leader/follower behavior of
fibroblasts and distinct tumor cell subpopulations can be tracked
within the invasive strands. We describe how to assess the role of
CAFs in the collective invasion of epithelial cancer cells. Analysis of
spheroid invasion patterns in 3D revealed that CAFs positioned at
the leading front within the invading strands and triggered cancer
cell dissemination of otherwise poorly invasive epithelial cancer cells
through specific cell–cell contacts [11]. Together with other studies, these findings highlight the importance of developing models
of tumor invasion that integrate cancer cell heterogeneity and
tumor–stroma interactions.
2 Materials
2.1 Cell Culture
All cell culture procedures should be performed under sterile conditions in a biological safety cabinet class II. In the following
protocol, human CAFs and HCC1806 cells were used as a model
for collective cell invasion but all the steps can be adapted to
different types of fibroblast and cancer cell if spheroid formation
and coculture are feasible (see Note 1).
1. Cells: Human Invasive Ductal Breast Carcinoma Cancer Associated Fibroblasts were collected from the BCI Breast Tissue
bank and immortalized by pBABE-Hygro-HTERT retroviral
transfection (Courtesy of Eric Sahai, Francis Crick Institute,
UK). HCC1806 cells (Human breast ductal cancer cell line).
2. Culture medium: Dulbecco’s modified Eagle’s medium
(DMEM) high glucose, supplemented with 10% fetal bovine
serum (FBS), 1% insulin-transferrin-selenium, 100 U/mL penicillin, 100 μg/mL streptomycin, and 29.2 mg/mL glutamine.
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Sefora Conti et al.
invasion where distinct cell populations interact, resulting in
enhanced invasiveness and survival of the moving cell cluster [5–
7]. Cancer Associated Fibroblasts (CAFs) are one of the most
abundant cell types in solid tumors, and they play an active role in
promoting tumor invasion and metastasis. Recent studies reported
that CAFs drive the initiation of collective dissemination of epithelial tumors through ECM remodeling and intercellular interactions
mediated by adherent junction proteins [8, 9]. These findings
highlight that the stromal contribution to metastasis is not limited
to chemical crosstalk but can also be mediated by direct mechanical
interactions [9, 10].
Here we provide a three-dimensional spheroid invasion assay to
assess the invasive pattern of cocultured CAFs and cancer cells in
organotypic ECM. This method enables multicellular coculture
and is compatible with time-lapse fluorescent imaging using
spinning disc microscopy. Collective invasion patterns can be monitored over a period of 24–72 h and leader/follower behavior of
fibroblasts and distinct tumor cell subpopulations can be tracked
within the invasive strands. We describe how to assess the role of
CAFs in the collective invasion of epithelial cancer cells. Analysis of
spheroid invasion patterns in 3D revealed that CAFs positioned at
the leading front within the invading strands and triggered cancer
cell dissemination of otherwise poorly invasive epithelial cancer cells
through specific cell–cell contacts [11]. Together with other studies, these findings highlight the importance of developing models
of tumor invasion that integrate cancer cell heterogeneity and
tumor–stroma interactions.
2 Materials
2.1 Cell Culture
All cell culture procedures should be performed under sterile conditions in a biological safety cabinet class II. In the following
protocol, human CAFs and HCC1806 cells were used as a model
for collective cell invasion but all the steps can be adapted to
different types of fibroblast and cancer cell if spheroid formation
and coculture are feasible (see Note 1).
1. Cells: Human Invasive Ductal Breast Carcinoma Cancer Associated Fibroblasts were collected from the BCI Breast Tissue
bank and immortalized by pBABE-Hygro-HTERT retroviral
transfection (Courtesy of Eric Sahai, Francis Crick Institute,
UK). HCC1806 cells (Human breast ductal cancer cell line).
2. Culture medium: Dulbecco’s modified Eagle’s medium
(DMEM) high glucose, supplemented with 10% fetal bovine
serum (FBS), 1% insulin-transferrin-selenium, 100 U/mL penicillin, 100 μg/mL streptomycin, and 29.2 mg/mL glutamine.
244
Sefora Conti et al.