Characterization of EpCAM in thyroid cancer biology by three-dimensional spheroids in vitro model
Creators
-
1.
Istituto Auxologico Italiano
-
2.
University of Milan
Description
Thyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs. The characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages’ inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay. Variable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient’s samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs. Our data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.
Notes (English)
File 1 and 2 represent the raw data necessary for the generation of the results described in Figure 1 of the manuscript, specifically the quantification of Western Blot experiments performed on tissues (file 1) and on immortalized cell lines (file 2).
File 3, 4 and 5 represent the raw data necessary for the generation of the results described in Figure 2. File 3 describes the quantification of the area of 3D spheres; file 4 and file 5 show the raw data calculated by ELDA online tool.
File 6 and 7 show the raw data necessary for the generation of the results described in Figure 3, specifically the quantification of Western Blot experiments performed on FRO-derived 3D spheres vs. FRO adherent cells (file 6), and the quantification of the 3D spheres obtained after treating FRO adherent cells with inhibitors of EpCAM cleavage and with DIP (file 7).
File 8, 9 and 10 show the raw data necessary for the generation of the results described in Figure 4 A, B, C (respectively). In particular, file 8 describes the results of the proliferation assay after treatment with Vemurafenib in FRO adherent cells and 3D spheres. File 9 describes the results of the proliferation assay after treatment with Vemurafenib in HTCC3 adherent cells and 3D spheres. File 10 describes the results of the proliferation assay after treatment with Vemurafenib in FTC133 adherent cells and 3D spheres.
File 11 and 12 represent the raw data necessary for the generation of the results described in Figure 4 D and E. In particular, file 11 describes the quantification of Immunofluorescence experiments in FRO adherent cells treated with Vemurafenib (column graph). File 12 describes the quantification of Immunofluorescence experiments in HTCC3 adherent cells treated with Vemurafenib (column graph).
File 13 and 15 represent the raw data necessary for the generation of the results described in Figure 4 D and E. In particular, file 13 shows the quantification of nucleus circularity analyzed in FRO adherent cells (boxplot graph). File 14 reports the quantification of nucleus circularity analyzed in HTCC3 adherent cells (boxplot graph).
File 14 and 16 represent the raw data necessary for the generation of the results described in Figure 4 D and E. In particular, file 14 shows the quantification of the Aspect Ratio of cell morphology analyzed in FRO adherent cells (boxplot graph). File 16 shows the quantification of the Aspect Ratio of cell morphology analyzed in HTCC3 adherent cells (boxplot graph).
File 17 and 18 show the raw data necessary for the generation of the results described in Additional File 1, Figure S1. Specifically, the quantification of Western Blot experiments performed on transfected FRO (file 17) and on transfected HEK293T (file 18).
File 19 represents the raw data necessary for the generation of the results described in Additional File 1, Figure S2. Specifically, the quantification of Western Blot experiments performed on FRO adherent cells treated with EGF and DIP.
Files
Files
(302.5 kB)
| Name | Size | Download all |
|---|---|---|
|
md5:2cfe81e0e6e4d68926cc1308b191aacd
|
15.2 kB | Download |
|
md5:3c2e69c587ac991608904fd4289eb403
|
20.3 kB | Download |
|
md5:acba8dea28f7e28d6042fb5739ff3a01
|
15.8 kB | Download |
|
md5:51b33d3e5368b3299907892c68275af4
|
20.7 kB | Download |
|
md5:5f27f142783144754856a2d39015ca78
|
22.1 kB | Download |
|
md5:9f65f799b3b58143151483ac5655cd63
|
19.7 kB | Download |
|
md5:9e45c1fcc1ebb37a1ce4afba30aa7b48
|
21.5 kB | Download |
|
md5:bc75075ed5b77a936dd844e4bc11c42a
|
17.7 kB | Download |
|
md5:a3cc443e5986fdc3feb298a1b0fcaad4
|
16.9 kB | Download |
|
md5:33c7d1bf778d8dceecbd0fc0d95e32f6
|
13.4 kB | Download |
|
md5:47b24e6e5af717fa8a4b98ed935abdeb
|
14.7 kB | Download |
|
md5:fbdd3a3271cabf8de5583bca8ac206ab
|
16.1 kB | Download |
|
md5:18f1fc0a5e116d4618084be788727822
|
9.9 kB | Download |
|
md5:36d3127c100ed2e8cff0169180415529
|
11.7 kB | Download |
|
md5:929ab98b5fc8e441f484f1977c0b49b6
|
11.7 kB | Download |
|
md5:1d38757c9095fbeb4977723ff28d6acf
|
14.2 kB | Download |
|
md5:f8ed25bc3b01f8d58b9951104530ea01
|
12.2 kB | Download |
|
md5:41a3fcf74f71139ca336a10f0e018964
|
14.5 kB | Download |
|
md5:3e5e4b7768f087445f5126652ad3c2ac
|
14.3 kB | Download |