Establishment of genetically diverse patient-derived xenografts of colorectal cancer

Abstract

Preclinical compounds tested in animal models often show limited efficacy when transitioned into human clinical trials. As a result, many patients are stratified into treatment regimens that have little impact on their disease. In order to create preclinical models that can more accurately predict tumor responses, we established patient-derived xenograft (PDX) models of colorectal cancer (CRC). Surgically resected tumor specimens from colorectal cancer patients were implanted subcutaneously into athymic nude mice. Following successful establishment, fourteen models underwent further evaluation to determine whether these models exhibit heterogeneity, both at the cellular and genetic level. Histological review revealed properties not found in CRC cell lines, most notably in overall architecture (predominantly columnar epithelium with evidence of gland formation) and the presence of mucin-producing cells. Custom CRC gene panels identified somatic driver mutations in each model, and therapeutic efficacy studies in tumor-bearing mice were designed to determine how models with known mutations respond to PI3K, mTOR, or MAPK inhibitors. Interestingly, MAPK pathway inhibition drove tumor responses across most models tested. Noteworthy, the MAPK inhibitor PD0325901 alone did not significantly mediate tumor response in the context of a KRAS(G12D) model, and improved tumor responses resulted when combined with mTOR inhibition. As a result, these genetically diverse models represent a valuable resource for preclinical efficacy and drug discovery studies.

Keywords: AZD8055; BEZ235; PD0325901; Targeted therapies; colorectal cancer; patient-derived xenograft; translational models.

Figure 1

PDX model characterization. A. Schematic representation of workflow in PDX establishment. B. Representative H&E images from randomly selected models illustrated clinical hallmarks of colorectal cancer. These included evidence of gland formation (i), mucin producing cells (ii), and columnar epithelium (iii). Scale bars indicate 50 μm. C. Using isolated tumor DNA from all models, eighty-five mutations (x axis) were profiled on the somatic mutation PCR panels directed against common mutations in APC, BRAF, CTNNB, FBXW7, KRAS, PIK3CA, SRC, and TP53. Each detected mutation is indicated as a peak along with gene symbol, coding change, and mutation.

Figure 2

In vivo efficacy of molecularly targeted compounds in three PDX models of CRC. (A-C) Three tumors with differing PI3K and RAS mutations were subcutaneously implanted into gender matched athymic nudes. Tumors were measured over time with treatment enrollment occurring randomly once tumor volumes reached 400 mm³. Compounds were administered as delineated in Table 4. The models associated with each panel are as follows: (A) COL02, (B) COL18, and (C) REC12. (D-F) At the conclusion of these studies, REC12 tumors from vehicle treated controls were harvested and sections were stained for pERK (D), pS6 (E), and pAKT (F) to determine the extent of MAPK, mTOR, and PI3K signaling within this model.

Figure 3

PI3K/mTOR/MEK combination therapy promotes statistically significant reductions in tumor burden. (A) Tumor growth of the REC12 PDX model was plotted to offer specific comparisons between single agents and combination therapies after 21 days. Significance was determined by repeated measures analyses and shown in panels (C and D). (B) Tumor volume averages from each treatment group were calculated at days 0 and 21 and presented as percentages of vehicle. (C, D) 95% confidence intervals for treatment groups were constructed and plotted following fit to a mixed linear model.

Figure 4

Combined therapy translates to a decrease in proliferation rate. Tumor sections from the indicated treatment groups of the REC12 model were probed with antibodies to Ki67, a marker of proliferation. Shown are representative images obtained at 100x magnification with insets depicting the most intense areas of staining within the field. Scale bar indicates 50 μm. Abbreviations: AZD + PD = AZD8055 and PD0325901. BEZ + PD = BEZ235 and PD0325901.

Figure 5

Effects of combined therapy on PI3K/mTOR/MAPK activation. Sections from vehicle and both combination regimens were stained for PI3K (pAKT, left panel), mTOR (pS6, middle panel), and MAPK (pERK, right panel) activation by immunohistochemistry. Scale bars indicate 50 μm. Abbreviations: AZD + PD = AZD8055 and PD0325901. BEZ + PD = BEZ235 and PD0325901.