Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model

Abstract

The potential utility of circulating tumor cells (CTCs) to guide clinical care in oncology patients has gained momentum with emerging micro- and nanotechnologies. Establishing the role of CTCs in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of CTCs for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. Recent attempts in the literature to culture CTCs isolated from metastatic patients using monoculture have had limited success rates of less than 20%. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully expanded CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis.

Figure 1. Overall strategy

The first step is to capture CTCs by flowing patient blood sample through a CTC-capture chip. The second step is to introduce fibroblasts and extracellular matrix (ECM) to the same chip to establish a co-culture environment for ex-vivo expansion of CTCs. The third step is to release and recover CTCs from device and the fourth step is downstream characterization.

Figure 2. Cancer cell capture and expansion on chip

(A) H1650 cell capture efficiency is determined (n = 3 for each condition). (B) Captured H1650 cells stained for cytokeratin 7/8 (green), CD45 (red) and DAPI (blue). (C) 100 H1650-GFP cells are captured and cultured in different environments (n = 3 for each condition). (D) 20–40 A549-GFP cells are captured and cultured in triplicate (indicated as Exp 1, 2 and 3). The curves characterize the growth of the cancer cells. (E) Scanning electron microscope (SEM) image of fibroblasts in gel cultured on chip. (F) EdU proliferation assay performed on cancer cells co-cultured with fibroblasts on chip. Green: cancer cells; Red: nucleus of proliferating cells; (G) Released H1975 lung cancer cells in a well-plate after on-chip culture. TTF-1(cyan), DAPI (blue), Cytokeratin 7/8 (red), Fibroblasts-GFP (green).

Figure 3. CTC capture and expansion data from patient samples

(A) Number of CTCs captured from 1 mL of blood prior to expansion. (left) healthy controls (n = 7), mean = 0.6; (right) patients (n = 19), mean = 4. Dotted line indicates the threshold value 2 CTCs/mL. CTC positive samples are determined as >2 CTCs/mL (n = 13). Images to the right are patient CTCs captured in the devices. CK7/8 (red), CD45 (green). (B) Growth chart comparing the number of CTCs captured on day 0 (blue columns) and the number of CTCs on day 14 (red columns) after expansion. CTCs are expanded successfully from 14 out of 19 patients. (C) After expansion, CTCs are characterized in well-plates with CK7/8 (red) surrounded by GFP-fibroblasts. (D) (left) CTCs from one patient sample (C23) are stained for TTF-1 (red). (right) CTC spheroids are formed in a 3D gel assay. (E) CTCs sorted out from fibroblasts stained positive for EGFR (cyan) and pan-CK (red) and are negative for FITC suggesting elimination of GFP-fibroblasts.

Figure 4. mRNA expression level in primary tumor and CTCs

(A to D). Cytokerain8 (CK8), cytokeratin18 (CK18), TTF-1 and EGFR gene expression level normalized to GAPDH. Tumor and CTCs mRNA from each patient sample are examined and compared. For example, “C20_T” represents patient C20 tumor (blue column) and “C20_CTC” represents patient C20 expanded CTCs (red column). The positive control is expanded H1650-GFP cells after initially spiking in blood with 100 cells, labeled as “H1650 (co)” (green column). The negative control is one healthy control as “HC” (purple column). (E). Invasion assay performed on three CTC samples, fibroblasts-GFP and H1650 cells for 24 hours. Representative images of the transwell membrane are shown on the right.

Figure 5. Sequencing data from patient samples

One TP53 point mutation (G to A) is found matched between primary lung tumor and cultured CTCs in patient C25. Another matched point mutation (G to A) is observed between primary tumor and CTCs in patient C26. Healthy controls showed no mutations. The table lists TP53 mutations found in CTCs and corresponding primary tumors in all 15 lung cancer patients tested.

Figure 6. Next-generation sequencing after targeted exon enrichment

(A) 8 paired tumor and CTC samples (C32-C39) plus one healthy control and one pure fibroblasts-GFP cell line are sequenced for 124 genes listed in the Qiagen Generead comprehensive cancer panel. Variants in each sample are identified by examining them in genome browser and confirmed by their absence in controls. (B) Four matched mutations are listed with their locations on exons, base change and amino acid change.