Figure 1: Expression of CD44v in metastatic breast cancer cells with lung colonization potential.

(a) IntegriSense fluorescence images of a representative mouse injected with 4T1 cells (1×10⁵) orthotopically into the mammary gland. Left colour bar and associated numbers indicate fluorescence intensity levels, with red and blue representing high and low fluorescence signals, respectively. LM, lung metastasis; PT, primary tumour. (b) RT–PCR analysis of total RNA from the indicated cell lines with primers targeted to exons 5 and 16 of the mouse CD44 gene as well as to the mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene (internal control). M, DNA size marker. (c) Flow cytometric analysis of CD44 isoform expression on the indicated cell lines with antibodies that recognize all isoforms of CD44 (pan-CD44) or only CD44v. Similar results were obtained in three independent experiments. (d) IntegriSense images of primary tumours formed as a result of orthotopic injection of sorted CD44v⁺ or CD44v⁻ 4T1 cells (1×10⁵) into the mammary gland of mice (upper). Tumour volume at 30 days after cell injection was calculated as length×(width)²/2 (bottom), and data are means±s.d. for eight animals per group. NS, not significant. (e) IntegriSense images of lung metastases formed in mice injected as in (d), and quantitative analysis of the total fluorescence intensity per lung metastatic lesion. Quantitative data are means±s.d. for 5 animals injected with CD44v⁺ cells or CD44v⁻ cells. **P<0.01 (Student's t-test). (f) Haematoxylin-eosin staining of lung metastases formed 30 days after orthotopic injection of CD44v⁺ or CD44v⁻ cells into the mammary gland of recipient mice. The boxed regions in the upper panels are shown at higher magnification in the lower panels. Arrows indicate metastatic nodules in the lung; asterisks indicate blood vessels; T, tumour. Scale bars, 300 μm (upper) or 100 μm (lower). (g) Flow cytometric analysis of CD44 isoform expression on tumour cells (6-TG-resistant and lineage marker-negative (Lin⁻) cells) isolated from lung metastases at 30 days after intravenous injection of CD44v⁻ 4T1 cells (1×10⁴) or both CD44v⁺ and CD44v⁻ 4T1 cells (5×10³ cells each).