Establishment and characterization of a carcinoma-associated fibroblast cell line derived from a human salivary gland adenoid cystic carcinoma

Figures & data

Figure 1. Establishment of CAF-SA. (A) Left: A single cell in a well (white arrows). Middle: Expanded cells. Right: Subcultured CAF-SA. Scale bar =500 μm. (B) The biomarker expression of CAF-SA. Cell were negative for Pan CK and α-SMA and positive for VIM, FSP-1 and FAP. Scale bar =50 μm.

Table 1. STR profiling of CAF-A1 and CAF-SA.

Marker	CAF-A1	CAF-SA
D5S818	10, 12	10, 12
D13S317	8, 9	8, 9
D7S820	11, 12	11, 12
D16S539	11, 12	11, 12
VWA	17, 18	17, 18
TH01	7, 9	7, 9
AMEL	X, X	X, X
TPOX	8, 10	8, 10
CSF1PO	11, 12	11, 12
D12S391	22, 24, 25	22, 25
FGA	22, 22	22, 22
D2S1338	23, 24	23, 24
D21S11	30.2, 30.2	30, 30
D18S51	14, 14	14, 19
D8S1179	14, 16	14, 15, 16
D3S1358	15, 17	15, 17
D6S1043	13, 19	13, 19
PENTAE	10, 13	10, 13
D19S433	13, 14.2	13, 14.2
PENTAD	9, 15	9, 15

Figure 2. CAF-SA promoted the migration and invasion of SACC cells. (A,B) Wound-healing assay. CAF-SA-CM promoted SACC-83 (A) and SACC-LM (B) migration significantly more than the control medium. (*p < .05. Scale bar = 200 μm) (C) SACC invasion assay using Transwell plates. CAF-SA-CM promoted SACC-83 and SACC-LM invasion significantly more than the control medium. (**p < .01. Scale bar = 200 μm). (D) SACC invasion assay using a microfluidic model. CAF-SA-CM promoted SACC-83 and SACC-LM invasion significantly more than the control medium. (**p < .01. Scale bar = 100 μm).

Figure 3. Clonogenic assay and tube formation assay. (A) Clonogenic assay. Representative images of colonies stained with crystal violet. (B) Quantification of clonogenic assay. CAF-SA-CM promoted SACC-83 and SACC-LM colony formation significantly more than the control medium. (*p < .05, **p < .01). (C) Tube formation assay. CAF-SA-CM promoted tube formation greatly. Scale bar = 100 μm.

Figure 4. CAF-SA promotes SACC growth in nude mouse models. (A) Photos of SACC-LM xenografts with or without CAF-SA. (B) Tumor volume analysis. CAF-SA-CM promoted SACC-LM growth significantly at Week 4, compared to controls (n = 5, two transplanted sites/nude mouse, *p < .05). (C) Typical xenografts of SACC-LM transplanted with or without CAF-SA after Ki67 immunostaining. Scale bar = 50 μm. (D) Quantification of Ki-67 staining. Positive cells significantly increased in the SACC-LM + CAF-SA group, compared to the SACC-LM group. (**p < .01).