CD200-expressing human basal cell carcinoma cells initiate tumor growth

Abstract

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Fig. 1.

Human BCC expresses hair follicle differentiation-specific keratins. (A) Human BCC. (B) Histology section showing interconnected islands of relatively monomorphic darkly stained BCC tumor cells. (C) RT-PCR with equal amounts of cDNA from hair follicle-rich scalp tissue and two different BCC samples (BCC1 and BCC2) showing expression of GAPDH and hair-specific keratins representing distinct layers of hair follicle differentiation, including ORS, companion layer, IRS, cuticle, and matrix. (D) Double-label immunofluorescence characterizes keratinocyte populations in hair follicle (Upper) and BCC (Lower), using K14 labeling to contrast expression of suprabasal ORS (K16 and K7), companion (K75), and IRS (K28) layer keratins. (Scale bars: 100 µm.)

Fig. 2.

The hair follicle KSC marker CD200 identifies a subpopulation of BCC tumor cells. (A) Double-label immunofluorescence of CD200 (red) together with suprabasal ORS K17 (green) expression. (Lower: Higher-magnification view.) Nonspecific CD200 labeling is seen within the nonviable inner aspect of the hair follicle adjacent to the hair shaft. (B) CD200 (green) expression by a small subset of basal and immediately adjacent suprabasal BCC tumor cells within K14-labeled (red) tumor nodules. The panel for each fluorescence label is shown, along with a merged image (Lower). (C) Representative flow cytometric analysis of human BCC sample cell suspensions labeled with isotype controls (Left) and with CD45 and CD200 (Right) showing the CD200⁺ CD45⁻ subpopulation (3.94%) of interest as well as CD200⁺ CD45⁺ (0.19%) cells. (D) Human BCC tumor sections labeled by immunohistochemistry demonstrate expression of GLI1, GLI2, and K17. (E) Three different BCC samples flow-sorted for CD200⁺ CD45⁻ and CD200⁻ CD45⁻ tumor subpopulations were compared with the GLI1-overexpressing cell line SJSA-1 (ATCC), three different BCC tissue samples (BCC1, BCC2, BCC3) and hair follicle-rich scalp tissue (HF). Expression of downstream hedgehog signaling targets GLI1, PDGFRα, and K17 was assessed by RT-PCR. GAPDH was used as internal cDNA control. (Scale bars: 100 µm.)

Fig. 3.

CD200⁺ BCC cells demonstrated increased colony-forming efficiency. (A) Unsorted BCC cells formed large and small tightly packed adherent spheroidal colonies when plated onto irradiated NIH 3T3 murine embryonic fibroblast feeder layers. (B) RT-PCR of SJSA-1, fresh human BCC tumor tissue (BCCt), cultured BCC cells (BCCc), and NIH 3T3 fibroblast cDNA demonstrating expression of hedgehog-regulated genes using human specific primers. Human- and murine-specific GAPDH primers were used to determine the relative contributions of human vs. murine cell cDNA in the BCC cell sample. (C) Tissue sections of xenografted BCC colonies reveal tumor nodules with H&E staining and immunohistochemistry reveal tumor nodules after 12 wk in vivo. (D) Colony forming efficiency was used to estimate the relative TIC frequency within 10⁵ cells from CD200⁺ CD45⁻ vs. CD200⁻ CD45⁻ vs. unsorted populations from five different BCC tumor samples. (Scale bars: 100 µm.)

Fig. 4.

Development of an in vivo BCC growth assay. (A) Schematic of the in vivo TIC assay, including the creation of a humanized stromal bed and etoposide pretreatment. When ≥3 × 10⁶ unsorted BCC cells were implanted, reproducible in vivo BCC growth was achieved (B). In vivo BCC growth was dependent on the dose of unsorted BCC cells implanted (C). Active in situ hedgehog signaling was confirmed by histology and immunohistochemistry (D). (Scale bars: 100 µm.)

Fig. 5.

The CD200⁺ CD45⁻ BCC cell subpopulation contains TICs. (A) Flow-sorted CD200⁺ CD45⁻ and CD200⁻ CD45⁻ subpopulations from 14 different fresh BCC samples were grafted in varying numbers. The CD200⁺ CD45⁻ subpopulation grafts (n = 38) gave rise to reproducible BCC growth with as few as 10⁴ cells implanted, whereas no growth was observed in grafts from the CD200⁻ CD45⁻ subpopulation (n = 14) when as many as 3 × 10⁶ cells were implanted. (B) In all cases, BCC growth was confirmed by histology and verified by immunohistochemistry to demonstrate active hedgehog signaling and differentiation. (Scale bars: 100 µm.)