Figure 6. Lkb1^fl/fl;Pten^fl/fl Lung SCC Contained SCA1⁺NGFR⁺ TPCs that Could Serially Transplant Squamous Disease.

(A) Representative flow cytometry plots for NGFR and SCA1 expression within the indicated EpCAM⁺CD45⁻CD31⁻ dissociated tumor cell populations. LP tumor cells showed much higher expression of both SCA1 and NGFR than either the Kras or Kras;p53 tumors.

(B) Quantification of SCA1- and NGFR-expressing cells with the EpCAM⁺CD45⁻CD31⁻ population as assessed by flow cytometry. The percentage of SCA1⁺NGFR⁺ in LP tumors is much higher than in Kras or Kras;p53 tumors: n = 23 for Kras tumors, n = 25 for Kras;p53 tumors, and n = 34 for Lkb1;Pten tumors (p < 0.0001).

(C) Representative immunohistochemical staining for NGFR on mouse SCC and ADC (a) and human SCC nodules (b). NGFR staining is strongly positive on SCC tumors but negative on ADC tumors. In the Lkb1;Pten;p53 tumors, distinct areas of ADC and SCC were adjacently located. NGFR staining was restricted to the SCC area (c). Scale bars represent 50 μm for (a) and (b) and 200 μm for panel (c).

(D) Quantification of tumorspheres derived from SCA1⁺NGFR⁺, SCA1⁻NGFR⁺, SCA1⁺NGFR⁻, and SCA1⁻NGFR⁻ FACS purified cells that were cocultured in Matrigel with equal amounts of CD45⁺CD31⁺ cells from the same primary tumors. Each fraction was seeded at 5,000 tumor cells/well. The colony-propagating ability of the SCA1⁺NGFR⁺ fraction in LP tumors is higher than that of the other fractions (p = 0.0011).

(E) Quantification of tumor propagation ability of FACS-isolated SCA1⁺NGFR⁺, SCA1⁺NGFR⁻, and SCA1⁻NGFR⁻ LP tumor cells. The secondary tumors were derived from intratracheal transplantation, with tumor formation latency of ~30 to 40 weeks. The tertiary tumors were derived from intrathoracic injection, with tumor formation latency of ~20 to 30 weeks. Ten thousand sorted cells from each fraction were injected for each fraction and each experiment. Only SCA1⁺NGFR⁺ populations could form tumors and be serially transplanted (p = 0.001 for secondary tumors, p = 0.002 for tertiary tumors, Fisher’s exact test).

(F) Representative immunohistochemical staining on tertiary tumors derived from SCA1⁺NGFR⁺ LP tumor cells after intrathoracic injection. The tumors retained a squamous histology and were positive for all of the squamous markers examined. Scale bars represent 100 μm for all panels.

Data are presented as mean ± SEM in (B) and (D). See also Figure S6.