Development of new preclinical models to advance adrenocortical carcinoma research

Abstract

Adrenocortical cancer (ACC) is an orphan malignancy that results in heterogeneous clinical phenotypes and molecular genotypes. There are no curative treatments for this deadly cancer with 35% survival at five years. Our understanding of the underlying pathobiology and our ability to test novel therapeutic targets has been limited due to the lack of preclinical models. Here, we report the establishment of two new ACC cell lines and corresponding patient-derived xenograft (PDX) models. CU-ACC1 cell line and PDX were derived from a perinephric metastasis in a patient whose primary tumor secreted aldosterone. CU-ACC2 cell line and PDX were derived from a liver metastasis in a patient with Lynch syndrome. Short tandem repeat profiling confirmed consistent matches between human samples and models. Both exomic and RNA sequencing profiling were performed on the patient samples and the models, and hormonal secretion was evaluated in the new cell lines. RNA sequencing and immunohistochemistry confirmed the expression of adrenal cortex markers in the PDXs and human tumors. The new cell lines replicate two of the known genetic models of ACC. CU-ACC1 cells had a mutation in CTNNB1 and secreted cortisol but not aldosterone. CU-ACC2 cells had a TP53 mutation and loss of MSH2 consistent with the patient's known germline mutation causing Lynch syndrome. Both cell lines can be transfected and transduced with similar growth rates. These new preclinical models of ACC significantly advance the field by allowing investigation of underlying molecular mechanisms of ACC and the ability to test patient-specific therapeutic targets.

Keywords: Lynch syndrome; adrenocortical carcinoma; cell lines; hyperaldosteronism; patient-derived xenografts.

Figure 1

Generation of ACC pre-clinical models. A. CU-ACC1 and B. CU-ACC2 nude mouse PDXs at the time of harvesting of the first passage. C. The appearance of CU-ACC1 cell line at passage 4 (10×) (upper panel) and passage 11 (10×) (lower panel) D. Species identification by genomic DNA approach confirming human origin (upper panel), and no 3T3-GFP tagged contamination is present in CU-ACC1 cells (lower panel) E. The appearance of CU-ACC2 cell line at passage 4 (10×) (upper panel) and passage 11 (10×) (lower panel) F. Species identification by genomic DNA approach confirming human origin (upper panel), and no 3T3-GFP tagged contamination is present in CU-ACC2 cells (lower panel)

Figure 2

Immunohistochemistry of human CU-ACC1 tumor and CU-ACC1 PDX. A. and B. The left columns show CU-ACC1 human tumor sample and the right two columns are from CU-ACC1 PDX. A. The immunochemistry stains include H&E, SF1, α-inhibin, Melan-A, Ki-67 and B. β-catenin, p53 and MSH2 C. Immunocytochemistry for Ki67 and SF1 (right columns) for CU-ACC1 cells (DAPI – left column)

Figure 3

Immunohistochemistry of human CU-ACC2 tumor and CU-ACC2 PDX. A. and B. The left columns shows CU-ACC2 human tumor sample and the right two column is from CU-ACC2 PDX. A. The immunochemistry stains include H&E, SF1, α-inhibin, Melan-A, Ki-67 and B. β-catenin, p53 and MSH2 C. Immunocytochemistry for Ki67 and SF1 (right columns) for CU-ACC2 cells (DAPI left column).

Figure 4

Whole exome sequencing of preclinical ACC models. A. Exome sequencing analysis of the human tumor sample and models revealed evidence of a CTNNB1 mutation in CU-ACC1 tissues and a TP53 mutation in CU-ACC2 tissues. Mutated gene allele frequencies were higher in the models compared to primary human tumors showing enrichment for the mutated clones. B. Sashimi plot of the CU-ACC2 human blood (H blood) confirms the heterozygous germline loss of MSH2 exons 1–6, with a complete loss of heterozygosity in the human tumor (H tumor), PDX and cell line.

Figure 5

RNA sequencing data analysis of preclinical ACC models. Left upper panel: Principle component analysis (PCA) of RNA sequencing profiles of normal adrenal compared with ACC cell lines and PDX models. Right upper panel and lower panels: Samples of normal adrenal (n=6), H295R cells (n=1) and newly developed models (CU-ACC1 and CU-ACC2 cells n=1 each, PDX n=2 each) were analyzed for transcript expression by RNA sequencing. Select transcript expression log2 levels are shown for adrenal specific markers (SF1, STAR, CYP11B1, CYP11B2, AGTR1, MCR2, and MRAP) as well as some of the known dysregulated genes in ACC (IGF2, BUB1B, PINK, CTNNB1).

Figure 6

LCMSMS analyzed steroid secretion profiles of CU-ACC1, CU-ACC2 and H295R ACC cell lines. Adrenal steroid secretome was analyzed at baseline (DMSO), and after ACTH 100nM, angiotensin 100nM and forskolin 10µM treatment for 24hrs.

Figure 7

Newly developed cell lines proliferate. A. Compared to H295R, both CU-ACC1 and CU-ACC2 cells show lower rates of proliferation. B. Doubling times for the ACC cell lines were determined. H295R cells have a doubling time of 25hrs, CU-ACC1 35hrs and CU-ACC2 29hrs.