Fig. 1.

Melanoma lesions harbor melanoma cells, which coexpress CD20 and HMW-MAA. (A) Melanoma cell suspensions were obtained from cutaneous melanoma biopsies, stained for CD20, HMW-MAA, tyrosinase (tyr), S100, Melan-A, and melanotransferrin (mtf), respectively, and analyzed by flow cytometry. No CD20⁺ cells were detected in biopsy from patient 5. NMM, nodular malignant melanoma; UCM, unclassified melanoma; SSM, superficial spreading melanoma. (B, Left) Cryostat sections of cutaneous melanoma lesions from patient 1 were stained for HMW-MAA using the PE-conjugated anti–HMW-MAA antibody EP-1 (red), for tyrosinase using the antityrosinase mAb T311, and for CD20 using the anti-CD20 mAb L27, respectively, followed by incubation with the biotinylated antimurine IgG₁ antibody and streptavidin-conjugated Alexa Fluor 488 (green). Staining with isotype-matched PE-conjugated antimurine antibodies were used as controls (Insert). Cell nuclei were visualized by DAPI staining (blue). (Right) Coexpression of CD20 and HMW-MAA was detected by the anti-CD20 mAb L27 and the anti–HMW-MAA mAb 763.74 and visualized by the Alexa Fluor 488 (green) and Alexa Fluor 555 (red) conjugated secondary antibodies, respectively. Photographs were merged using Adobe Photoshop software. (Scale bars, 20 μm.) (C) Melanoma cells from patient 1 biopsy were cultivated in adherent cultures or grown to melanospheres in human embryonic stem cell medium as described (21). Spheroidal and adherent cells were analyzed for expression of CD20 and HMW-MAA by flow cytometry using the PE-conjugated anti–HMW-MAA mAb EP-1 and the FITC-conjugated anti-CD20 mAb L27. (D) RT-PCR analysis for CD20 mRNA expression of adherently growing melanoma cells (number of passages > 50) and of melanosphere cells derived from the same biopsy. A 459-bp fragment was derived from CD20 mRNA and a 267-bp fragment from β-actin mRNA as control. No CD20 mRNA was detected in cells from patient-5 biopsy.