Precision Microfilters as an all in one System for Multiplex Analysis of Circulating Tumor Cells

Abstract

Enumeration of circulating tumor cells (CTCs) from cancer patient blood is an established diagnostic assay used to evaluate patient status as a singleplex test. However, in the coming age of personalized medicine, multiplex analysis of patient CTCs, including proteomic and genomic techniques, will have to be integrated with CTC isolation platform technologies. Advancements in microfabrication have demonstrated that CTCs can be isolated and analyzed using microfluidic lab-on-a-chip devices. However, to date, most microfluidic devices are either still in the development phase, not applicable to all clinical tests, or are not commercially available. To overcome these discrepancies, we describe an all-in-one device for the isolation and multiplexing of clinically applicable CTC assays. Microfilters present an ideal lab-on-a-chip platform for analysis of CTCs as non-toxic and inert materials allow for a multitude of tests from cell growth through clinical staining techniques, all without background interference. Lithographically fabricated microfilters, can be made with high porosity, precise pore dimensions, arrayed pore distribution, and optimized for CTC size-based isolation. In this study we describe microfilter use in isolation and in situ analysis of CTCs using multiple sequential techniques including culture, FISH, histopathological analysis, H&E staining, photobleaching and re-staining. Further, as a proof of principle, we then describe the ability to quantitatively release patient derived CTCS from the microfilters for potential use in downstream genomic/proteomic analysis.

Figure 1. Overview of the work flow and methodologies described in this study

(A) Assays are developed and optimized with cancer cell lines spiked into normal blood samples (Green). Cancer cell lines are spiked into blood samples collected in CellSave® tubes. The sample is filtered and CTCs are identified using presence of anti-cytokeratin and anti-EpCAM, with absence of anti-CD45. CTCs are quantified, then stained by FISH, H&E, etc. (B) Assays are run on patient samples (Purple). Blood from cancer patients were collected in CellSave® tubes, filtered and CTCs were identified. CTCs were counted and the clinically useful subtypes were quantified (i.e. CTCs in Division, apoptotic CTCs, etc). Many of these cells were further subtyped by FISH or H&E stain (Figure 2). (C). Proof of principal assay for expansion of CTCs. Viable cancer cell lines were spiked into normal blood collected in either EDTA and isolated by filtration. The filter bound cells were then expanded in culture media for eventual use in other models^,. Dotted arrow indicates that cell lines were used. Solid lines indicate the assay was developed with cell lines and has proceeded onto patient samples.

Figure 2. Flow through device with all-in-one reaction chamber

(A) The microfilter chip device consists of a holder and removable microfilter. (B) The device is designed with a reaction chamber which can be used run assays without then need to transfer the cells. (C+D) The entire device connects to sterile disposable syringes and a medical pump which allows uniform flow through the filter.

Figure 3. Isolation, Culture and expansion of Cells isolated on CellSieve^™

(A) Live MCF-7 cells spiked into vacutainers, isolated by filtration and grown on the filter for 2–3 weeks. The 3 dimensional clustering attributed to this cell line can be seen on the filter. (green=anti-cytokeratin, blue=DAPI) (B) Live PANC-1 cells spiked into vacutainers, isolated by filtration and grown on the filter for 2–3 weeks. This cell line can be seen growing as a monolayer on the filter. (C) SKBR3 cancer cell line is spiked into blood collected by CellSieve^™. The CTCs are identified using presence of anti-cytokeratin and anti-EpCAM, with absence of anti-CD45. After CTCs are counted the cells are subtyped by HER2 FISH. (D) Live SKBR3 cells spiked into vacutainers, isolated by filtration and grown on the filter for 2–3 weeks. The expanded colonies can be directly analyzed as a whole colony, or as individual cells, molecularly by HER2/CR17 FISH analysis. (E) After filtration, a single CTC can be identified and harvested using a micropipette (F) Removal of a single cell for downstream analysis (i.e. whole genome amplification, mRNA analysis). (G) After filtration, cells can be identified with histopathological stains (e.g. H&E) for cytological analysis or (H) After H&E, external cell structures can be analyzed by SEM.

Figure 4. Cytological analysis and subtyping of CTCs from patients

(A) CTC from a breast cancer patient categorized as early apoptotic with punctate cytokeratin, and an nucleus that appears as malignant with an abnormal salt-and-pepper pattern. This CTC subtype is associated with a favorable outcome ^, . (B) CTC from a breast cancer patient categorized as late apoptotic with punctate cytokeratin and a nucleus which also appears punctate, or blebbing. This CTC subtype is associated with a favorable outcome. (C and D) CTCs from 2 breast cancer patients in the final stages of division (i.e. telophase/cytokinesis). This CTC subtype is associated with poor prognosis. Scale=30 μm box.

Figure 5. Bleaching and restaining CTCs

After identifying and imaging patient derived CTCs using epithelial cell markers (e.g. cytokeratin+, EpCAM+ and CD45−), the PE fluorescence from EPCAM was bleached, freeing the channel for an additional marker. The CD45 was negative, allowing the channel to remain open for an additional marker. The DAPI channel and the Cytokeratin-FITC channel remained unchanged, and can be used to identify the CTCs after restaining. The sample was then restained with the mesenchymal marker Vimentin with efluor660 and a stromal regulation marker CXCR4 with PE. Scale=72 μm box.

Figure 6. CTC release efficiency from microfilters for downstream analysis

To determine the ability of the CellSieve^™ filters to backwash cells off the filters. MCF-7 cells (~50 cells) were spiked into 7.5mL normal blood and filtered. Cells were then backwashed off the filters and counted, as was the number of both CTCs and WBCs remaining on the filter. Y-axis shows the percentage of cells captured on the second filters versus cells still remaining on the first filters. Black bar, MCF7 cells backwashed off the first filter and captured on the second filter versus cells remaining on the first filter. Striped bar= normal WBCs backwashed off the first filter and captured on the second filter versus cells remaining on the first filter. White bar, 16 patient samples of CTCs processed as described for the MCF-7 cells. Striped bar, same 16 patient samples of WBCs backwashed off the filter versus remaining on the filter as described above.