Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes

Abstract

Background: Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth.

Methods: To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models. We identified high and low NE subtypes of SCLC in all of our sample types, and characterized their properties.

Results: We found that 16% of human SCLC tumors and 10% of SCLC cell lines were of the low NE subtype, as well as cell lines from the GEM model. High NE SCLC lines grew as non-adherent floating aggregates or spheroids while Low NE lines had morphologic features of the variant subtype and grew as loosely attached cells. While the high NE subtype expressed one of the NE lineage master transcription factors ASCL1 or NEUROD1, together with NKX2-1, the entire range of NE markers, and lacked expression of the neuronal and NE repressor REST, the low NE subtype had lost expression of most NE markers, ASCL1, NEUROD1 and NKX2-1 and expressed REST. The low NE subtype had undergone epithelial mesenchymal transition (EMT) and had activated the Notch, Hippo and TGFβ pathways and MYC oncogene . Importantly, the high and low NE group of SCLC lines had similar gene expression profiles as their SCLC tumor counterparts.

Conclusions: SCLC tumors and cell lines can exhibit distinct inter-tumor heterogeneity with respect to expression of NE features. Loss of NE expression results in major alterations in morphology, growth characteristics, and molecular properties. These findings have major clinical implications as the two subtypes are predicted to have very different responses to targeted therapies.

Keywords: Small cell lung cancer (SCLC); epithelial-mesenchymal transition; genetically engineered mouse model; neuroendocrine tumors; tumor heterogeneity.

Figure 1

Generation of a 50-gene NE signature. Left panel: the top 25 genes overexpressed in NE cell lines and the top 25 genes overexpressed in non-NE cell lines were selected from a volcano plot (the colors of the dots are shown on a gradient scale from blue to red in proportion to the distance between each point and the origin (0, 0). Red dots, highly significant). Right panel: the heat map shows that these 50 genes separate lung NE from non-NE lines. NE, neuroendocrine.

Figure 2

Evaluation of the NE gene signature in various public and private datasets of NE and other lung tumors. The score ranges from −1.0 to 1.0. Positive score predicts for NE while negative score predicts for non-NE. Multiple platforms (RNAseq, Affymetrix, Illumina and custom gene expression microarray) were used. GEO accession numbers are shown when available (17,19,26,27). NE, neuroendocrine.

Figure 3

Heterogeneity of NE scores in SCLC tumors and cell lines. The distribution of NE scores in SCLC tumors and cell lines is displayed. Samples whose values were below that of 1 standard deviation (1 SD) were regarded as having low or negative scores, with the remainder having high NE scores. NE, neuroendocrine; SCLC, small cell lung cancer.

Figure 4

Cell culture and cell line xenograft appearances of classic (NCI-H128) and variant (NCI-H82) forms of SCLC. (A) Classic cell line NCI-H128 has a high NE score, grows as floating spheroids (illustrated) or clusters of non-adherent cells. (B) Variant cell line NCI-H82, having MYC gene amplification and over expression, grows as loosely attached cells, either singly or in small aggregates, sometimes in single cell rows (“Indian file” pattern). (C) Xenograft formed after subcutaneous inoculation of NCI-H128 in an immunosuppressed mouse shows relatively small cells with indistinct cell borders, and nuclei having a “salt and pepper” distribution of chromatin and relatively small nucleoli. These appearances are typical for those of SCLC (classic morphology). (D) NCI-H82 cell line xenograft shows somewhat larger loosely aggregated cells with distinct cell borders, larger nuclei with single prominent nucleoli and peri-nucleolar chromatin clearing (variant morphology). (A,B) Phase contrast appearance of living cultures photographed by inverted microscope; (C,D) tissue section of xenograft tumor, formalin fixed, paraffin embedded, stained with hematoxylin and eosin.

Figure 5

Appearances of dual negative cell lines and resultant cell line derived tumors from the Myc driven GEM model (11) (also see text). (A) Phase contrast image of cell line derived from culture of an original tumor after an in vitro culture period greater than 14 days. The cells grow as relatively large, loosely attached floating cells or as cells partially attached to the substrate. There is a tendency to grow in single cell rows (“Indian file” pattern). (B) Histological appearances of a subcutaneous tumor after inoculation of the cultured tumor cells. While some cells resemble the classic form of SCLC (arrow), most of the cells are larger than those of classic SCLC, and have one or more prominent nucleoli with paranucleolar chromatin clearing. While the appearances have some resemblance to the human variant form of SCLC, the greater intercellular heterogeneity more closely resembles the appearances of the large cell (undifferentiated) form of human NSCLC. Formalin fixed, paraffin embedded section, stained with hematoxylin and eosin. SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer.

Figure 6

Expression of NE transcription factors ASCL1 and NEUROD1 in SCLC tumors and cell lines. In both tumors and cell lines, four RNA expression subtypes can be identified (upper panel): (I) ASCL1 high, (II) NEUROD1 high, (III) dual high and (IV) dual low. The expression values of ASCL1 and NEUROD1 (FPKM) were log2 transformed. The dual low subtype was associated with a low NE score (lower panel). ***, P<0.001 (t-test). NE, neuroendocrine; SCLC, small cell lung cancer.

Figure 7

Expression of NE transcription factor ASCL2 in SCLC tumors. Left panel, the correlation between ASCL2 expression and ASLC1 expression in SCLC tumors. Right panel, ASCL2 expression in NE high and NE low SCLC tumors. The expression values of ASCL1 and ASCL2 (FPKM) were log2 transformed. NE, neuroendocrine; SCLC, small cell lung cancer.

Figure 8

Examples of positive and negative correlations between the NE scores and expression of indicated genes. Pearson correlation coefficient was calculated in 81 SCLC tumors. The expression values of the genes (FPKM) were log2 transformed. NE, neuroendocrine; SCLC, small cell lung cancer.

Figure 9

Summary of the main differences between NE high and low subtypes of SCLC. The NE high subtype usually has classic SCLC morphology, a high NE cell score with high expression of ASCL1 and most or all NE markers, expression of NKX2-1, the Notch inhibitors DLL3 and DLK1, and an epithelial phenotype. NEUROD1 expression is more variable. By contrast, the low NE phenotype has low or absent expression of ASCL1 and NEUROD1 (“dual low”), low or absent expression of NE cell markers, activation of Notch, HIPPO and TGFβ pathway genes and profound epithelial to mesenchymal (EMT) transition resulting in a mesenchymal phenotype. REST, an inhibitor of NE differentiation, is expressed while MYC is often over-expressed. Some but not all dual negative cases express ASCL2, possibly acting as an alternative driver transcription factor. Many dual negative tumor cells exhibit the variant form of SCLC. NE, neuroendocrine; SCLC, small cell lung cancer.