KRAS and PIK3CA mutation frequencies in patient derived xenograft (PDX) models of pancreatic and colorectal cancer are reflective of patient tumors and stable across passages

One key obstacle to the translation of advances in cancer research into the clinic is a deficiency of adequate preclinical models that recapitulate human disease. The development and application of validated preclinical models that reflect patient histological, cellular, and molecular characteristics is needed. Current preclinical models rely heavily on conventional cell line xenograft models which are established by engrafting human tumor cell lines cultured in the laboratory into mice. This model is widely acknowledged to provide useful, but unreliable predictive capacity for anti-tumor activity in humans (Sharpless and Depinho 2006). One possible explanation for the unreliability of cell line xenograft results translating to the clinic, is that these cells represent clonal tumor populations that have selectively grown on plastic and have adapted to growth outside of the natural tumor microenvironment (Frese and Tuveson 2007; Tentler et al. 2012). Because cell line xenograft models lack stromal cells, which are increasingly recognized as a critical element for tumorigenesis, these models fail to accurately recapitulate tumor biology and tumor response to therapy (Bhowmick et al. 2004; Sharpless and Depinho 2006; Frese and Tuveson 2007)

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens

Irreversible JNK1-JUN inhibition by JNK-IN-8 sensitizes pancreatic cancer to 5-FU/FOLFOX chemotherapy

Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors

CTC isolation using a microfluidic chip.

<p>(A) Design of the CTC microfluidic chip with sinusoidally shaped capture channels and brightfield images of (B) the capillary tube inserted into the on-chip entry channel where whole blood enters the microfluidic chip, (C) sinusoidally shaped capture channels where anti-human EpCAM antibodies are immobilized for CTC capture, (D) exit channel, and (E) impedance sensor with two Pt electrodes located adjacent to the exit channel to detect released CTCs. (F) Cells captured from whole blood of PDX-tumor bearing mice visualized directly on the microfluidic chip following immunostaining with DAPI (blue), human cytokeratin 8/19 (CK, red), and mouse CD45 (green). The staining pattern of DAPI-positive, human CK-positive, and mouse CD45-negative is characteristic of human CTCs. (G) A rare contaminating mouse leukocyte bound non-specifically to the microfluidic chip stained DAPI-positive, human CK-negative, and mouse CD45-positive. Contaminating leukocytes were excluded from enumeration due to the high specificity of the electrical impedance detector for cancer cells.</p

Tumor volume and CTC burden response to treatment.

<p>(-) No data due to microfluidic chip failure.</p

CTC enumeration from PDX-tumor bearing mice.

<p>CTCs captured and enumerated from whole blood of non-tumor and tumor bearing PDX mice. Five non-tumor bearing mice were analyzed for their CTC level (median  = 0 CTCs/250 µL, range  = 0–1 CTCs/250 µL), while CTCs were enumerated from 31 of 31 PDAC PDX mice (median  = 11 CTCs/250 µL, range  = 1–83 CTCs/250 µL) (p = 0.0008, Wilcoxon).</p

Tumor growth inhibition with BKM120 treatment.

<p>BKM120 treatment of PDAC PDX mice for 28 days inhibited tumor growth (Mean fold change, FC  = 1.56; SEM  = 0.148; n = 9) compared to vehicle (mean FC  = 2.16; SEM  = 0.221; n = 8, p = 0.0185, t-test).</p

Response of CTC burden to BKM120 treatment.

<p>PDAC PDX mice were treated with vehicle or BKM120 for 28 days. CTCs were enumerated from whole blood on day 0 prior to the first treatment and on day 28 after the last treatment. CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (pre-treatment, pre: median  = 26.61 CTCs/250 µL, range  = 7–63 CTCs/250 µL, n = 8; post-treatment, post: median  = 2.21 CTCs/250 µL, range  = 0–79 CTCs/250 µL, n = 8; p = 0.0207, Wilcoxon) while no significant change was observed in the vehicle group (pre: median  = 23.26 CTCs/250 µL, range  = 4–43 CTCs/250 µL, n = 4; post: median  = 11.89 CTCs/250 µL, range  = 6–146 CTCs/250 µL, n = 8; p = 0.8081, Wilcoxon) One post BKM120 treatment sample had no detectable CTCs and is not plotted on scale.</p

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens