Primary tumor genotype is an important determinant in identification of lung cancer propagating cells

Abstract

Successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor. Therapeutic advances therefore necessitate the characterization of the cells that are able to propagate a tumor in vivo. We show an important link between tumor genotype and isolation of tumor-propagating cells (TPCs). Three mouse models of the most common form of human lung cancer each had TPCs with a unique cell-surface phenotype. The cell-surface marker Sca1 did not enrich for TPCs in tumors initiated with oncogenic Kras, and only Sca1-negative cells propagated EGFR mutant tumors. In contrast, Sca1-positive cells were enriched for tumor-propagating activity in Kras tumors with p53 deficiency. Primary tumors that differ in genotype at just one locus can therefore have tumor-propagating cell populations with distinct markers. Our studies show that the genotype of tumor samples must be considered in studies to identify, characterize, and target tumor-propagating cells.

Figure 1

Orthotopic transplantation of Kras and Kras;p53-flox tumor cells recapitulates the primary tumor phenotype. (A) Cartoon of the transplantation scheme used to assay lung tumor-propagating cells (TPCs) through serial transplantation. (B) H&E staining of primary (left) and secondary (right) Kras lung adenocarcinomas showing similar pathological grade, nuclear features, and general tumor architecture. (C) H&E staining of primary (left) and secondary (right) Kras;p53-flox lung tumors showing similar histopathological characteristics of advanced adenocarcinoma, including pleomorphic nuclei and rare giant cells. All images, 200× magnification. Scale bar = 100μM.

Figure 2

Sca1 + cells from Kras;p53-flox tumors are lung tumor-propagating cells. (A) Representative FACS analysis of Kras;p53-flox tumor cells used for transplantation (top). Limiting dilution transplantation of the sorted cells indicated that the Sca1 + population was significantly enriched for TPCs (table). (B) Sca1- cell transplants yielded smaller, more diffuse lesions (left), whereas Sca1 + cell transplants yielded secondary tumors that recapitulated the histopathology of primary Kras;p53-flox tumors (right, compare to Figure 1C). (C) Immunofluorescence (IF) staining with antisera raised against SP-C (red), CCSP (green), and counterstain DAPI (blue) showed that primary Kras;p53-flox lung adenocarcinomas (left) are mainly composed of SP-C+ cells, a pattern recapitulated in secondary tumors from Sca1 + cell transplants (right). (D) Serial transplantation of secondary tumor cell populations revealed a lack of tumor formation from Sca1 - cells (left), in contrast to tertiary tumor development from Sca1 + cells (right). All images, 200× magnification. Scale bar = 100μM. See also Figure S1 and Table S1 for additional data.

Figure 3

The Sca1 + and Sca1- populations from Kras lung tumors are equally capable of propagating tumors. (A) Representative FACS analysis of Kras tumors showing a pattern of Sca1 staining similar to Kras;p53-flox tumors (top, compare to Figure 2A). Limiting dilution transplantation indicated that Sca1+ and Sca1- Kras tumor cells are identical in tumor-propagating potential (table). (B) Secondary tumors from Sca1- (left) and Sca1+ (right) Kras tumor cell transplants displayed similar pathological features. (C) IF analysis of secondary tumors (as in Figure 2) revealed similar SP-C and CCSP marker status in secondary tumors from Sca1- cells (left) and Sca1+ cells (right). (D) Both Sca1- Kras tumor cells (left) and Sca1+ tumor cells (right) were capable of serial transplantation to form tertiary tumors. All images, 200× magnification. Scale bar = 100μM. See also Figure S2 and Table S2 for additional data.