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1 Supporting Information CD200 Expressing Human Basal Cell Carcinoma Cells Initiate Tumor Growth Chantal S Colmont 1, Antisar BenKetah 1, Simon H Reed 2, Nga Voong 3, William Telford 3, Manabu Ohyama 4, Mark C Udey 5, Carole L Yee 5, Jonathan C Vogel 5, Girish K Patel 1.

2 Colmont CS et al, 2012 Supplemental Figure 1 B A Hair follicle: K14 & K19 D C BCC: K14 & K19 BCC: K14 & K16 E BCC: K17 & K75 BCC: K17 & K16

3 Supplemental Figure 1 Colmont CS et al, 2012 F

4 Supplemental Figure 1 Colmont CS et al, 2012 Fig. 1. Human BCC express hair follicle differentiation specific keratins within the inner cell mass. (A) K19 is expressed in hair follicle basal and suprabasal ORS keratinocytes below the level of the hair follicle bulge. (B) In BCC K19 expression in restricted to small populations within the tumor inner cell mass. Likewise, (C) K16 is normally expressed by suprabasal ORS keratinocytes and K75 (D) which is restricted to the companion layer keratinocytes are also restricted to the inner cell mass of BCC tumors. Although K17 (D & E) is also expressed by hair follicle suprabasal ORS keratinocytes, in BCC its expression is throughout the tumor. (F) Hair follicle keratin expression was observed by immunofluorescence in all BCC with each tumor exhibiting varying levels of differentiation similarly in all tissue section labelled.

5 Colmont CS et al, 2012 Supplemental Figure 2 A Hair follicle BCC Isotype control B Ki67 Isotype control Bcl-2 Isotype control CD24 C CD71 CD146 CD200

6 Supplemental Figure 2 Colmont CS et al, 2012 Fig. 2. Cell surface markers used to enrich human hair follicle bulge keratinocyte stem cells define subpopulations in human BCC. (A) CD24 was expressed on differentiated suprabasal ORS keratinocytes, as well as the BCC inner cell mass. (B) The transferrin receptor CD71 was expressed by basal cells of both hair follicles and BCC nodules in keeping with its role in cell proliferation. Cell surface glycoprotein MUC18 CD146, was expressed by hair follicle basal keratinocytes below the level of the bulge, but expression was restricted to endothelial cells in BCC tumor samples. In contrast CD200, the hair follicle bulge keratinocyte stem cell marker, was expressed by a small population of hair follicle ORS cells and also occasional BCC basal layer cells. Higher magnification images are shown in the inserts. Consistent with the pattern of inward differentiation observed with expression of hair follicle keratins and CD24, (B) cell proliferation assessed by Ki67 labeling and expression of the anti-apoptotic protein BCL-2 (C) were mostly expressed by basal layer BCC cells.

7 Colmont CS et al, 2012 Supplemental Figure 3 A Isotype BCC cell suspension labelled with Dapi & pan-cytokeratin Isotype Isotype CD200+ CD45- D Isotype 7-AAD EpCAM CD200- CD45 cells labelled with Dapi & pan-cytokeratin CD200+ CD45 cells labelled with Dapi & pan-cytokeratin C CD200- CD45- CD200 EpCAM B Dissociation remnants CD200 BCC pre-dissociation 7-AAD EpCAM CD45

8 Supplemental Figure 3 Colmont CS et al, 2012 E SJSA-1 cells CD200+ CD45 Normal keratinocytes F CD200+ CD45- Merge Gli1 Pan-cytokeratin DAPI CD200- CD45-

9 Supplemental Figure 3 Colmont CS et al, 2012 Fig. 3. Isolation and analysis of BCC subpopulations. (A) Mechanically and enzymatically digestion of BCC tumor tissue was optimised using dissociation remnant histology, resulting in cell suspensions containing pan-cytokeratin positive tumor keratinocytes. After flow sorting, both CD200+ CD45- and CD200- CD45- BCC tumor subpopulations were greatly enriched for pan-cytokeratin positive tumor keratinocytes. (B) Many BCC tumor samples contained within them EpCAM positive BCC keratinocytes, after dissociation flow cytometric analysis revealed ~90% live single cells using the viability dye 7-amino-actinomycin D (7-AAD), with ~62% EpCAM (CD326) positive cells (red arrow). (C) EpCAM positive BCC cells contained a small subpopulation of EpCAM+ CD200+ cells, highlighted with the red arrow. (D) Flow sorting of CD200+ CD45- and CD200- CD45- BCC tumor subpopulations achieved 86% and 98% purities when reanalysed by flow cytometric analysis. (E) Compared to the positive control SJSA-1 cell line and normal human keratinocytes, the CD200+ CD45- BCC keratinocytes expressed Gli1 as a marker of active hedgehog signalling. (F) Primary human BCC tumor tissue was dissociated and labelled with propidium iodide to determine the DNA content by flow cytometric analysis of CD200+ CD45- and CD200 CD45- flow sorted sub-populations was used to determine the percentage of cells undergoing cell division (in G2-M), 7.3% versus 4.6% respectively.

10 Supplemental Figure 4 Colmont CS et al, 2012 A B CD200+ CD45- BCC Colonies CD200- CD45- BCC Colonies Fig. 4. BCC tumor cells form colonies in vitro. When plated onto an irradiated 3T3 feeder layer BCC cells from fresh tumors form spheroidal colonies that are adherent to the 3T3 cells. (A) The frequency of colony formation was dependent upon the number of unsorted BCC cells plated. (B) Sorted BCC cells, CD200+ CD45- and CD200- CD45- subpopulations, also gave rise to colonies in vitro. However the frequency of colonies and their size differed, CD200+ CD45- cells gave rise to larger colonies that continued to grow.

11 Colmont CS et al, 2012 Supplemental Figure 5 A 0 of 27 0 of 8 6 of 7 B 1 Human BCC Xenograft BCC Gli 1 K17

12 Supplemental Figure 5 Colmont CS et al, 2012 Fig. 5. BCC tumor tissue xenograft growth was dependent on the creation of a stromal bed and etoposide pre-treatment. (A) BCC tumor tissue (0.5cm 3 ) was implanted directly beneath the skin on the backs of athymic nude, SCID-beige and NOD-SCID mice. After 12 weeks graft sites were excised and histologic sections were examined for tumor growth, no tumor growth was observed in the 27 mice studied. We had previously observed that SCC growth was dependent on a stromal bed created by pre-implantation of a glass disk or gelfoam dressing. However this approach also failed to yield in vivo BCC tumor growth (0 of 8). Reproducible BCC growth (6 of 7) was only possible when mice were also pretreated with intraperitoneal etoposide prior to grafting. (B) BCC tumor tissue xenografts retained architectural similarity to the original BCC histology and maintained features of active hedgehog signalling: K17 and Gli1 expression.

13 Supplemental Figure 6 Colmont CS et al, 2012 Fig. 6. Flow sorted for CD200+ CD45- and CD200- CD45- tumor subpopulations were compared to the human embryonic stem tumor cell line NTERA2 (ATCC), three different BCC tissue samples (BCC1, BCC2, BCC3) and hair follicle rich scalp tissue (HF) by RT-PCR for expression of genes involved in embryonic stem cell self-renewal. GAPDH was an internal cdna control.

14 Supplemental Figure 7 Colmont CS et al, 2012 A B Media 0.1% DMSO Vis. 5µM Vis. 10µM Cyclop. 15µM Cyclop. 30µM Fig. 7. BCC colony forming efficiency is not affected by smoothend antagonists. Dissociated BCC tumor cells were plated at low density in triplicate in media, media containing 1% DMSO, varying concentrations if vismodegib (Vis.) and cyclopamine (cyclop.). The media was changed every three days and the treatment conditions maintained. The number of colonies in the centre of the 24-well plated were photographed (A) and counted (B) after 7 days using 4x magnification. To confirm the presence of human cells, cdna isolated from the cultures was amplified by PCR using human specific GAPDH primers.

15 Supplemental Table 1 History of BCC Colmont CS et al, 2012 * Mice were further immunocompromised at the time of grafting by splenectomy and intraperitoneal administration of anti-lymphocyte serum. ** Beige nude mice have fewer NK cells. *** Beige nude mice were grafted without undergoing splenectomy nor intraperitoneal administration of antilymphocyte serum.

16 Supplemental Table 1 Colmont CS et al, 2012 Pawlowski A, Haberman HF. Heterotransplantation of human basal cell carcinomas in "nude" mice. J Invest Dermatol. 1979; 72: Löning TH, Mackenzie IC. Immunohistochemical studies of basal cell carcinomas transplanted into nude mice. Arch Dermatol Res. 1986; 279: Stamp GW, Quaba A, Braithwaite A, Wright NA. Basal cell carcinoma xenografts in nude mice: studies on epithelial differentiation and stromal relationships. J Pathol. 1988; 156: Hales SA, Stamp G, Evans M, Fleming KA. Identification of the origin of cells in human basal cell carcinoma xenografts in mice using in situ hybridization. Br J Dermatol. 1989; 120: Grimwood RE, Johnson CA, Ferris CF, Mercill DB, Mellette JR, Huff JC. Transplantation of human basal cell carcinomas to athymic mice. Cancer. 1985; 56: Grimwood RE, Glanz SM, Siegle RJ. Transplantation of human basal cell carcinoma to C57/BALB/C bgj/bgj-nu/nu (beige-nude) mouse. J Dermatol Surg Oncol. 1988; 14: Grimwood RE, Tharp MD. Growth of human basal cell carcinomas transplanted to C57/Balb/C bgj/bgj nu/nu (beige-nude) mice. J Dermatol Surg Oncol. 1991; 17: Carlson JA, Combates NJ, Stenn KS, Prouty SM. Anaplastic neoplasms arising from basal cell carcinoma xenotransplants into SCID-beige mice. J Cutan Pathol. 2002; 29:

17 Supplemental Table 2 Colmont CS et al, 2012 Record of primary human BCC unsorted cell xenografts

18 Supplemental Table 3 Colmont CS et al, 2012 Limiting dilution analysis of unsorted cells in primary human BCC xenografts from different BCC samples

19 Supplemental Table 4 Colmont CS et al, 2012 Record of primary human BCC sorted CD200+ CD45 and CD200 CD45- cell xenografts

20 Supplemental Table 5 Colmont CS et al, 2012 Limiting dilution analysis of CD200+ CD45 sorted cells in primary human BCC xenografts from different BCC samples

21 Supplemental Table 6 Colmont CS et al, 2012 Primer sequences for keratin genes

22 Supplemental Table 7 Colmont CS et al, 2012 Primer sequences for hedgehog signalling and self renewal genes