Circulating tumor cells as a biomarker of response to treatment in patient-derived xenograft mouse models of pancreatic adenocarcinoma

Abstract

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.

Figure 1. CTC isolation using a microfluidic chip.

(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.

Figure 2. CTC enumeration from PDX-tumor bearing mice.

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).

Figure 3. Patient and PDX tumor.

H&E staining of the de-identified patient tumor (A, B) and corresponding PDX tumor (C, D). Both the patient and PDX tumors are consistent with PDAC with desmoplastic stroma (20× magnification).

Figure 4. Response of CTC burden to BKM120 treatment.

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.

Figure 5. Tumor growth inhibition with BKM120 treatment.

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).