Patient-derived xenograft (PDX) models of colorectal carcinoma (CRC) as a platform for chemosensitivity and biomarker analysis in personalized medicine

Abstract

Patient-derived xenograft (PDX) tumor models represent a valuable platform for identifying new biomarkers and novel targets, to evaluate therapy response and resistance mechanisms. This study aimed at establishment, characterization and therapy testing of colorectal carcinoma-derived PDX. We generated 49 PDX and validated identity between patient tumor and corresponding PDX. Sensitivity of PDX toward conventional and targeted drugs revealed that 92% of PDX responded toward irinotecan, 45% toward 5-FU, 65% toward bevacizumab, and 61% toward cetuximab. Expression of epidermal growth factor receptor (EGFR) ligands correlated to the sensitivity toward cetuximab. Proto-oncogene B-RAF, EGFR, Kirsten rat sarcoma virus oncogene homolog gene copy number correlated positively with cetuximab and erlotinib sensitivity. The mutational analyses revealed an individual mutational profile of PDX and mainly identical profiles of PDX from primary tumor vs corresponding metastasis. Mutation in PIK3CA was a determinant of accelerated tumor doubling time. PDX with wildtype Kirsten rat sarcoma virus oncogene homolog, proto-oncogene B-RAF, and phosphatidylinositol-4,5-bisphosphate 3-kinaseM catalytic subunit alfa showed higher sensitivity toward cetuximab and erlotinib. To study the molecular mechanism of cetuximab resistance, cetuximab resistant PDX models were generated, and changes in HER2, HER3, betacellulin, transforming growth factor alfa were observed. Global proteome and phosphoproteome profiling showed a reduction in canonical EGFR-mediated signaling via PTPN11 (SHP2) and AKT1S1 (PRAS40) and an increase in anti-apoptotic signaling as a consequence of acquired cetuximab resistance. This demonstrates that PDX models provide a multitude of possibilities to identify and validate biomarkers, signaling pathways and resistance mechanisms for clinically relevant improvement in cancer therapy.

Keywords: Colorectal carcinoma; In vivo drug testing; Patient-derived xenograft models; Personalized medicine.

Figure 1

Histological and mutational status of CRC PDX. (A) Staining of the original patient tissue paired to the corresponding PDX tissue. Representative patient/PDX pairs were chosen: Co10194 is derived from a primary colon carcinoma sample, Co9978 from a rectum sample, Co7809 was derived from a liver metastasis and Co7475 is derived from a lung metastasis. Upper panel, original patient tissue: HE staining; panel 2 to 4, PDX tissue: HE staining; IF staining for human nuclei, DAPI = blue; human nuclei = orange (Cy3); IHC staining for EpCAM. (B) Representative HE staining of consecutive passages of PDX Co5854 and Co11061 demonstrate characteristic phenotype of human colorectal carcinoma. Scale bar represents 100 µm. (C) Summary of mutational status of all PDX models.

Figure 2

Tumor doubling times (TDT) of the PXD models. The tumor doubling time was measured once the models were considered as stably established. The PDX with mutated PIK3CA are marked by an “*.”

Figure 3

Chemosensitivity of the PDX models. (A) Three representative chemosensitivity curves of PDX Co9587, Co9689B, and Co10383 are shown. Groups of 5 tumor-bearing mice were treated either with vehicle (control group) or specific drug as monotherapy. (*P< 0.05, **P < 0.01, ***P < 0.001). (B) Summary of sensitivity characteristics of the PDX models toward standard of care cytostatic and targeted drugs based on the optimal treated to control (optT/C) values, expressed in percent. (C) Sensitivity of PDX models toward cetuximab: the 49 PDX models are arranged according to their optT/C-value for cetuximab and are correlated to the KRAS mutational status. The bar below represents the relative tumor volume (RTV) values. The bar colors indicate the respective mutational status of KRAS. (D) Comparison of optT/C-values for EGFR-inhibitors cetuximab and erlotinib between PDX with wildtype KRAS, BRAF, and PIK3CA (triple wt) and PDX with an activating mutation in one of these genes (mut).

Figure 4

Molecular characterization of PDX regarding GCN and AREG/EREG protein expression and chemosensitivity toward targeted drugs. (A) GCN of EGFR in the 49 CRC PDX models, determined by a real-time PCR (left panel) and correlation to cetuximab response (right panel). (B) AREG expression at protein level (by ELISA) in the PDX models and correlation to cetuximab response (right panel). The protein concentrations have been normalized to the total protein content in the sample. Three PDX samples from different passages were measured (n = 3). (C) Summary of correlation analysis (spearman coefficients rs and their corresponding P values) of the GCN of the key molecules of the EGFR network (left table) and the sensitivity of the PDX toward cetuximab and erlotinib, as well as ligand expression at mRNA and protein level (right table).

Figure 5

Analysis of newly generated cetuximab resistant PDX models. (A) The PDX models Co7596 and Co10718 were initially cetuximab sensitive (left panels). After continued cetuximab treatment the cetuximab resistant models Co7596-cetux and Co19718_cetux were generated (right panels). (B) Comparison of genetic profiles in the 2 PDX pairs by the Illumina TruSeq Amplicon Cancer Panel reflected by mutated allele frequency and specific amino acid (AA) mutations. (C) Analysis of relative mRNA expression of EGFR, HER2, HER3, and HER4 in the PDX pairs. (D) Analysis of relative mRNA and protein expression of the EGFR ligands AREG, BTC, EGF, EREG, and TGFα in the PDX pairs. (*P< 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Gene expression and proteome, phosphoproteome analysis of cetuximab sensitive vs resistant PDX. (A) Comparison of the gene expression of EGFR receptors and molecules involved in EGFR signal transduction. (B) Heatmaps of significantly changed proteins and phosphosites (One-sample t test, adj. P value < 0.1, reproducibility filter = 0.01) within the EGFR, PI3K/AKT/Mtor, and apoptosis pathways (Wiki Pathway annotations) for the Co10718 model. P values are calculated with data of 2 replicates for the Co10718_cetux models normalized against Co10718_orig models. The annotation column shows 2 lanes for 2 replicates of sensitive (Co10718_orig) and cetuximab resistant (Co10718_cetux) models with green and magenta colors, respectively. On the heatmap, blue color indicates down-regulation whereas red color corresponds to proteins and phosphosites up-regulated in the cetuximab resistant comparing to sensitive pair. (C) Volcano plot of the P values vs the logFC (fold change) of enrichment scores of PTM-SEA (Ref DOI: 10.1074/mcp.TIR118.000943). Two-sample t test is performed between resistant and sensitive replicates of the Co10718 model. Proteins crossing the significance lines (|logFC| ≥ 2, Pvalue ≤ 0.05) are colored in blue or red. (D) Heatmap depicting enrichment scores of significantly regulated pathways in the PTM-SEA analysis.