Volume 114, Issue 6, Pages 1196-1205 (June 1998) Abnormal expression of CD44 variants in the exfoliated cells in the feces of patients with colorectal cancer  Takekazu Yamao*, Yasuhiro Matsumura*, Yasuhiro Shimada*, Yoshihiro Moriya‡, Ken-Ichi Sugihara‡, Takayuki Akasu‡, Shin Fujita‡, Tadao Kakizoe§  Gastroenterology  Volume 114, Issue 6, Pages 1196-1205 (June 1998) DOI: 10.1016/S0016-5085(98)70425-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Map of CD44 gene products, showing exons and primers used in this study. RT-PCR for each sample was conducted using Sp1 and Sp2 primers. The probes for CD44 standard, v6, and v10 were made by PCR using primers of SE×16 and AE×17, Sv6 and Av6, and Sv10 and Av10, respectively. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 RT-PCR–Southern blot analysis of fecal samples and immunohistochemical staining with monoclonal antibodies to total CD44 and CD44v6 protein in a surgically resected specimen in the same patient (patient 1 in Table 1). (A) Southern blot analysis of RT-PCR products from fecal samples using a CD44s exon–specific probe. Lane 1, fecal sample before surgical resection of the primary tumor; lane 2, fecal sample after surgical resection of the primary tumor; lane 3, resected normal colonic mucosa; lane 4, resected cancerous lesion. (B) Southern blot analysis of RT-PCR products from the fecal samples using a CD44v6-specific probe in the same patient. Lane 1, fecal sample before surgical resection of the primary tumor; lane 2, fecal sample after surgical resection of the primary tumor; lane 3, resected normal colonic mucosa; lane 4, resected cancerous lesion. Molecular weight markers a, b, c, d, and e denote 1.35-, 1.08-, 0.87-, 0.60-, and 0.31-kilobase pairs, respectively. Immunohistochemical detection of CD44 expression: (C) normal colonic mucosa stained for total CD44, (D) normal colonic mucosa stained for CD44v6, (E) colonic adenocarcinoma stained for total CD44, and (F) colonic adenocarcinoma stained for CD44v6. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A–P) Immunohistochemical staining with monoclonal antibodies to total CD44 and CD44v6 protein in surgically resected specimens from 4 patients with colorectal cancer (patients 2 [A–D], 3 [E–H], 4 [I–L], and 5 [M–P] in Table 1). (Q) RT-PCR–Southern blot analysis of exfoliated cells in feces before surgery (lanes 1, 3, 5, and 7) and after surgery (lanes 2, 4, 6, and 8) in each patient using a CD44s exon–specific probe (patient 2, lanes 1 and 2; patient 3, lanes 3 and 4; patient 4, lanes 5 and 6; and patient 5, lanes 7 and 8). (R) RT-PCR and Southern blot analysis of the same filter using a CD44v6-specific probe. (S) RT-PCR and Southern blot analysis of the same filter using a CD44v10-specific probe. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Southern blot analysis of RT-PCR products from individual samples of exfoliated cells in fecal samples from patients with colorectal cancer (patients 6–25 in Table 1). PCR products obtained with CD44 standard primers (Sp1 and Sp2, see Figure 1) were resolved on an agarose gel, and after Southern blotting, the filter was hybridized with a CD44-exon standard probe (see Materials and Methods). (A) Exfoliated cells in fecal samples from patients before surgical resection of the primary tumors. (B) Samples from the corresponding patients after surgical resection of the primary tumors. Molecular weight markers a, b, c, d, and e denote 1.35-, 1.08-, 0.87-, 0.60-, and 0.31-kilobase pairs, respectively. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 The same filter as in Figure 4 was rehybridized with a CD44v6-specific probe. (A) Exfoliated cells in fecal samples from patients before surgical resection of the primary tumors. (B) Samples from the same patients after surgical resection of the primary tumors. Molecular weight markers a, b, c, d, and e denote 1.35-, 1.08-, 0.87-, 0.60-, and 0.31-kilobase pairs, respectively. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 The same filter as in Figure 4 was rehybridized with a CD44v10-specific probe. (A) Exfoliated cells in fecal samples from patients before surgical resection of the primary tumors. (B) Samples from the same patients after surgical resection of the primary tumors. Molecular weight markers a, b, c, d, and e denote 1.35-, 1.08-, 0.87-, 0.60-, and 0.31-kilobase pairs, respectively. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Southern blot analysis of RT-PCR products from individual samples of exfoliated cells in fecal samples from 15 normal health volunteers. (A) Hybridization with a CD44s exon–specific probe. (B) Hybridization with a CD44v6-specific probe. (C) Hybridization with a CD44v10-specific probe. The filter used in each hybridization was identical. Gastroenterology 1998 114, 1196-1205DOI: (10.1016/S0016-5085(98)70425-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions