Simple and Convenient Method for the Isolation, Culture, and

Subscriber access provided by University of Winnipeg Library

Methods/Protocols

Simple and Convenient Method for the Isolation, Culture, and Recollection of Cancer Cells from Blood by Using Glass-Beads Filters Babita Shashni, Hidehiko Matsuura, Riku Saito, Takuma Hirata, Shinya Ariyasu, Kenta Nomura, Hiroshi Takemura, Kazunori Akimoto, Naoyuki Aikawa, Atsuo Yasumori, and Shin Aoki ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01335 • Publication Date (Web): 01 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Simple and Convenient Method for the Isolation, Culture, and Recollection of Cancer Cells from Blood by Using Glass-Beads Filters

Babita Shashni,a Hidehiko Matsuura,b Riku Saito,b Takuma Hirata,b Shinya Ariyasu,c Kenta Nomura,d Hiroshi Takemura,d,e Kazunori Akimoto,a,e Naoyuki Aikawa,b,c,e Atsuo Yasumori,b and Shin Aoki*,a,c,e,f

a

Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

b

Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Nijuku, Katsushika-ku, Tokyo 125-8585, Japan c

Center for Technologies against Cancer, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan d

Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

e

Division of Medical Science-Engineering Corporation, Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan f

Imaging Frontier Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

*Corresponding authors: E-mail, [email protected]

ACS Paragon Plus Environment

1

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 54

ABSTRACT Circulating tumor cells (CTCs) are tumor cells that originate from primary cancer tissues, enter the blood stream in the body, and metastasize to the other organs. Simple and convenient methods for their detection, capture, and recovery from the blood of cancer patients would be highly desirable. We report here on a simple and convenient methodology to trap, culture, and recollect cancer cells, the sizes of which are greater than that of normal hematologic cells by the use of glass-beads filters (GBF). We prepared GBFs with a diameter of 24 mm and a thickness of 0.4 mm and 1.2 mm with well-defined pores, by sintering round-shaped glass beads (diameter: 63–106 m). A small integrated glass-beads filter (iGBF) with a diameter of ca. 9.6 mm for the use in filtering a small volume of blood was also designed and prepared. Using GBF and iGBF, it was possible to efficiently capture mouse Lewis lung carcinoma cells expressing green fluorescent protein spiked in saline/blood by single and repeated (circulation) filtrations in in vitro experiments with very small amounts of red blood cells being captured. In addition, we successfully captured B16 CTCs from the blood of a B16 melanoma metastasis mouse model by iGBF. Cancer cells/CTCs captured on/in the GBF could be cultured and efficiently recovered from the filters. Filtration by GBF had negligible effect on the adherent and proliferative characteristics of cancer cells. Simple and convenient methods for the capture, culture and recollection of CTCs by GBF along with flexibility of GBF, which permits them to be molded into suitable architectures having the desired shape and size, offers good method for early and convenient diagnosis and treatment of cancer and related diseases.

Key words: Glass-beads filter, Filtration, Circulating tumor cells, Size based separation.


2

Page 3 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Circulating tumor cells (CTCs) are tumor cells that are found in the peripheral blood of cancer patients.1, 2 It is reported that they originate from the primary cancer tissues, travel around in the blood stream, and then are arrested at other organ sites distant from the original cancer sites, resulting in the metastatic colonization.3-5 It is assumed that such metastasis that causes 90% of cancer-associated human deaths could be detected, predicted, and/or managed if CTCs could be captured from the blood of cancer patients, analyzed, and used in drug testing.6,7 Currently used methods for detection and capture of CTCs from whole blood mainly include biological and physical methods.8-12 Biological approaches involve the capture of CTCs based on the expression of tumor specific antigens such as cytokeratins and epithelial cell adhesion molecules (EpCAM), etc.13-20 However, the non-uniform expression of CTC markers, due to cancer heterogeneity and during epithelial-to-mesenchymal transition renders biological based approaches for CTC detection and capture as a less reliable approach.21-24 On the other hand, physical methods for CTC capture are based on differences in biophysical deformities (shape, size and related characteristics) between CTC and hematologic cells.25-31 It has been reported that the sizes of tumor cells are larger than those of hematologic cells, suggesting that the use of a size-based method for the capture of CTC using filtration systems might be a more convenient approach than biological-based methods.32-40 In this context, the use of microdevices such as microfilters, microfluidics, microsieve chips, and micropinching chips have been reported for use in the size-based enumeration and enrichment of CTCs.39-48 Most of the microdevices require additional confirmation of the isolated CTCs by staining with CTC markers such as EpCAM (whose expression varies with biological process), which are analyzed by trained pathologists, and it is also not easy to recover viable CTCs after staining for CTC confirmation. Moreover, to our knowledge, there is no method that is able to treat a large volume (L~L order) of non-diluted and/or diluted blood samples which isolates live cancer cells. As such, convenient, rapid and cost-effective strategies for isolation and growth of CTC are highly required. ACS Paragon Plus Environment

3


Page 4 of 54

Herein, we report on a simple methodology for trapping, culturing, and recollecting cancer cells via the use of a glass-bead filter (GBF) having a diameter of 24 mm and thickness of 1.2 and 0.4 mm, and small integrated GBF (iGBF) with a diameter of ca. 9 mm and thickness of ca. 2 mm (Figure 1). GBFs were prepared by sintering (650–700C for 1 h) commercially available round-shaped glass beads (diameter: 63–106 m) to produce well-defined pores with an adequate porosity based on our assumption of the size threshold between cancer cells and hematologic cells (Figure S1 in Supporting Information).49 Namely, a suitable porous structure was constructed that allows smaller normal hematologic cells such as red blood cells (RBC) to pass through the filters, while larger cells such as cancer cells are trapped on/in the filters. Commercially available glass filters (GF) were also purchased and then mechanically shaved and polished to adjust their forms to the exact same size and shape as those of GBF and their cell filtration properties were compared. The entrapment of in vitro cancer cell lines and CTCs from an experimental model blood and from model mice using GBF and iGBF (and GF) by a single filtration assembly and repeated (circulated) filtration system is also described. Furthermore, we also report that the cancer cells that were captured on/in GBF and iGBF could be further grown and recollected by using the specific holder (designed especially for the culture and recollection of the trapped cancer cells).


4

Page 5 of 54

Trapped cancer cells

Blood + cancer cells

Culture of trapped cancer cells Cancer cells (Green fluorescent protein (GFP)-positive)

Incubation

Glass-beads filter (GBF) (Prepared from glass beads having diameter of 63-100 μm)

50 µm

50 µm

GBF

Culture

Diameter of GBF: 24 mm Thickness: 1.2 mm or 0. 4 mm

Rubber ring Filter assembly

Integrated glass-beads filter (iGBF) Space to hold blood sample 20 mm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Trapped large cancer cells

Analysis on particle size analyzer

GBF in a well or

Recovery, characterization, and analysis of cultured cells

iGBF in a well

GBF Glass tube 9.6 mm

Thickness: 2 mm

Figure 1. Schematic presentation of capture of cancer cells using the filtration system designed in this work. Glass-beads filter (GBF) and integrated GBF (iGBF) (in a dashed box) were designed and prepared to capture large cancer cells and grow them on/in GBF and iGBF.

■ MATERIALS Reagents. Cell culture. ・Trypsin-EDTA (Gibco, 25-200-056) ・Fetal bovine serum (Capricorn Scientific, Japan, FBS-12B) ・Benzyl penicillin potassium (Wako, Japan, 021-07732)


5


Page 6 of 54

・Streptomycin sulphate (Wako, Japan, 194-08512) ・ HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) (Dojindo Laboratories, Japan, GB10) ・1-Thioglycerol (Sigma-Aldrich, M4007)

・Sodium Chloride (Nacalai Tesque, Japan, 313-33)

Preparation of GBF, GBF-holder, and GBF-case. ・Round-shaped soda lime silicate glass beads with diameters of 63–106 m, 90~125 m, 70  5 m, and 100  5 m (Union Co. Ltd., Japan). ・ Polypropylene photopolymer material for the construction of GBF-holder (for CTC recovery by centrifuge) and that of GBF-case (for CTC trap in blood circulating system) (Stratasys Ltd., VeroClear (RGD810)) on 3D printer (Stratasys Ltd., Object pro 30). ・ Support material for the construction of GBF-holder (for CTC recovery by centrifuge) and that of GBF-case (for CTC trap in blood circulating system) (Stratasys Ltd., Support SUP705) on 3D printer (Stratasys Ltd., Object pro 30). ・Acetone (Nacalai Tesque, Japan, 12533-44) ・Ethanol (FUJIFILM WAKO Pure Chemical Corporation, Japan, 327-00027)

Equipment. ACS Paragon Plus Environment

6

Page 7 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


General. ・Lab coat ・Gloves ・Safety glasses

・Pipettes ・Tweezer/Forceps ・Clips ・Centrifuge tubes 50 mL (FastGene, FG200) ・Centrifuge tubes 15 mL (Watson, 233-150C) ・Centrifuge tubes 5 mL (Watson, 233-150C) ・Haemocytometer (Hirschmann Techcolor, Germany) ・Bright-field microscope (Olympus 1X70, Japan)

・Rotating polishing machine (Musashino-Denshi, Japan, MA-150) ・3D printer (Stratasys Ltd., Object pro 30) ・Particle size analyzer (JASCO-Occhio, Japan, IF nano-200)


7


Page 8 of 54

・Fluorescence microscopy (Keyence, Japan, Biorevo, BZ-9000) ・Micro syringe pump (AS ONE, Co. Ltd., Japan, SPE-1) ・UV-visible spectrophotometer (StellarNet Inc., FL, USA) ・UV-visible spectrophotometer (JASCO, Japan, V-550) ・A circulation (peristaltic) pump (EYELA, MP-4000) ・ Dialysis circulation tubes (for the dialysis of animals such as dogs and cats) for Mera crystal flow with a MCA dialysis filter series (Senko Medical Instrument Mfg. Co. Ltd., Japan). ・Centrifugation machine (Kubota, Japan, 5930) ・Carbon dioxide gas incubator (ESPEC CORP., Japan, BNA-111) ・A shaker to shake blood in the bottles during circulation filtration (IKA, Japan, IKA-VIBRAX-VXR)

・ Anesthesia machine for small animals compact type, anesthesia box, and anesthesia gas recovery equipment (Shinano factory, Japan, SN-487-0T, SN48785, and SN489-2) ・PC software, “Cancer Cell Finder (CCF) ver. 1.0”49

Preparation and Characterization of Glass-Beads Filter (GBF) and Integrated GBF (iGBF). ・A 24 mm diameter alumina (Al2O3) tube for GBF


8

Page 9 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


・A 9 mm glass tube for iGBF ・6 m and 15 m polystyrene beads (Polysciences, Inc., PA, USA) ・An electric furnace (Nitto Kagaku, Co. Ltd., Japan, NHK-170) ・A drying oven (Iuchi, Co. Ltd., Japan, DO-300)

Filters (or Mesh) to Compare Capture Efficiency Using Cancer Cells. ・・・・・

Commercially available glass filter (GF) (Shibata, Japan, D13000-419) Nylon mesh (Clever Co. Ltd., Toyohashi, Japan) Polystyrene mesh (Clever Co. Ltd., Toyohashi, Japan) Thimble filter (Advantec Toyo (Toyo Roshi Kaisha), Ltd., Tokyo, Japan) Fabric (non-woven) filter (Tapyrus Co. Ltd., Tokyo, Japan).

Cancer Cell Lines and Blood. ・Lewis lung carcinoma (LLC)-enhanced green fluorescent protein (EGFP) (LLC-EGFP) cell line ・B16 melanoma cell line ・SP2/O myeloma cell line ・MDA-MB 157 breast cancer cell line ・Bovine blood supplemented with sodium heparin (10000 U /1 L) (Domestic Animal Resource Development, Tokyo, Japan)

Culture of Cancer Cells, Their Capture, Growth, and Recovery by Means of GBF and iGBF. ACS Paragon Plus Environment

9


・High glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Japan, LS11995040) ・RPMI 1640 medium (Nacalai Tesque, Japan, 186-02155) ・Low glucose DMEM (Nacalai Tesque, Japan, 08456-65) ・12 well plates (Thermo Scientific, 150628) ・24 well plates (Thermo Scientific, 142475)

Animal Experiments. ・C57BL/6 mice (ca. 6-7 weeks old) (CLEA, Japan) ・Heparin (Mochida Pharmaceutical Co., LTD, Japan) ・21 G and 25 G needles (Terumo Corporation, Japan, NN-2138R and NN-2525R) ・1 mL Syringe (Terumo Corporation, Japan, 435SS-01T) ・Isoflurane as anesthesia (FUJIFILM Wako Pure Chemical Corporation, Japan, 099-06571) ・Mouse surgical equipment (scissors and forceps)

■ METHODS Preparation of Glass-Beads Filter (GBF).


10

Page 10 of 54

Page 11 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. To prepare GBF, round-shaped soda lime silicate glass beads (ca. 1 g) with diameters of 63–106 m were molded in a 24 mm diameter alumina (Al2O3) tube and a stainless weight was placed on the glass beads in an alumina tube (Figure S1a-1c in Supporting Information). 2. Heat treatment for sintering glass beads was carried out on an aluminum plate between two stainless plates (120 mm width ×120 mm depth ×10 mm height) under and above alumina tubes, as shown in Figure S2a in Supporting Information, at atmospheric pressure in an electric furnace. 3. Temperature in the electric furnace was raised from room temperature to 700 C at the increasing rate of 10 C /min and then kept for 1 h to produce a GBF with a diameter of 24 mm and a thickness of 1.2 mm and 0.4 mm (Figure S1a-1c in Supporting Information). 4. The temperature in the electric furnace was cooled down to room temperature. 5. The prepared GBFs was washed with acetone (ca. 20 mL for each GBF) and ethanol (ca. 20 mL for each GBF), dried completely at 60 C for over 12 h in a drying oven, immediately prior to use for the filtration of cancer cells.

The porosity of GBF was calculated from the GBF’s bulk density and apparent density. The bulk density was calculated from its bulk volume and weight, and the apparent density was obtained from the underwater mass and specific gravity of pure water (note that there are very few closed pores in the GBF). Apparent density was obtained by the following equation (1), in which W is the mass of GBF, W’ is the underwater mass of GBFs, and Wρ is specific gravity of water.

Apparent density= Wρ/(W-W')

(Archimedes' principle)

And then, porosity was calculated by the following equation (2)


11

(1)


Porosity

=

{1–(GBF's

apparent

density)/(GBF’s

Page 12 of 54

bulk

density)}x100

(%)

(2)

The density and porosity of the GBF were 1.7 g/cm3 and 30.1 %, respectively. For comparison, a commercially available glass filter (GF) (Shibata, Japan), whose diameter of 24 mm, were mechanically shaved and polished to adjust its thickness to 1.2 mm and diameter of 22.5~23.5 mm (without being sintered). Its density and porosity were 1.3 g/cm3 and 40.2 %, respectively.

Preparation of Integrated Glass-Beads Filter (iGBF). 1. Round-shaped soda lime silicate glass beads (ca. 0.15 g) with diameters of 63–106 m were molded in a glass tube with a diameter of 9 mm, in which a stainless rod was placed on the glass beads (Figure S1d in Supporting Information). 2. Two stainless plates (120 mm width ×120 mm depth ×10 mm height) were placed under and above the glass tubes, as shown in Figure S2b in Supporting Information. 3. Heat treatment for sintering glass beads was carried out at atmospheric pressure in an electric furnace, in which temperature was raised from room temperature to 670 C at the increasing rate of 10 C/min and then kept at 670 C for 1 h. 4. The temperature in the electric furnace was cooled down to room temperature. 5. iGBFs were washed with acetone (ca. 20 mL for each iGBF) and ethanol (ca. 20 mL for each iGBF). 6. iGBFs were heated again to 690 C at the increasing rate of 10 C/min again and then kept at 690 C for 1 h in the electric furnace. 7. After the temperature in the electric furnace was cooled down to room temperature, the prepared GBFs are washed with acetone (ca. 20 mL for each iGBF) and ethanol (ca. 20 mL for each


12

Page 13 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


iGBF), dried at 60 C for over 12 h in a drying oven, immediately prior to use for the filtration of cancer cells. The thickness of glass-beads filter part was measured to be ca. 2 mm.

Filtration of Polystyrene Beads Through GBF. To confirm the capture efficiency of GBF, preliminary filtration experiments were carried out using polystyrene beads (Figure S5 in Supporting Information). 1. Given polystyrene beads in water (polystyrene beads with a diameter of 6 m as a model of RBCs and those with a diameter of 15 m as a model for cancer cells, based on information from Figure S3 in Supporting Information) were filtered through a 1.2 mm GBF using a syringe pump with a flow rate of 4 mL/min (Figure S4a in Supporting Information). 2. The decrease in the number of polystyrene beads after filtration through the GBF was monitored by the change in the absorbance at 600 nm in UV-visible absorption spectra (StellarNet Inc., FL, USA) based on calibration curves of the absorbance at 600 nm (Figure S4b in Supporting Information). 3. The number of polystyrene beads that passed through the filters was evaluated (Figure S5 in Supporting Information) using the equation (3).

Passing ratio (%) = (absorbance after filtration/absorbance before filtration) x 100, where the absorbance

of

the

initial

sample

(before

filtration)

was

taken

as

100

%.

(3)

The movement of 15 m polystyrene beads through GBF and GF observed by high-speed camera is displayed in Supporting Movie S1 and S2 in Supporting Information, respectively.

Preparation of a GBF Holder (GBF-Holder) for Centrifugation-based Recovery of Cancer Cells


13


Page 14 of 54

Trapped on/in GBF and a GBF-Case for Cancer Cell Trap in a Blood Circulation System by 3D Printing. The GBF holder was made of a clear polypropylene photopolymer material and constructed with a 3D printer (Object pro 30, Stratasys Ltd.). The outside diameter (at the widest part) and the height of the GBF-holder were 25.2 mm and 14.7 mm, respectively, as shown in Figure S11a and S11b in Supporting Information. 1. The 3-dimensional computer-aid design (CAD) files (Standard Triangulated Language (STL) format) for a GBF-holder and a GBF-case were prepared based on the corresponding 2-dimensional blueprints as shown in Figure S11 and S13 in Supporting Information, respectively. 2. A box of polypropylene photopolymer material, VeroClear (RGD810), and that of support material, Support SUP705, were put in a 3D printer. 3. The aforementioned STL files were opened by the software “Object Studio” installed in the PC to control 3D printer and started the printing. 4. The raw products made from VeroClear and Support SUP705 were kept in water bath at 40~50 oC for a couple of days to roughly remove support material, which is soluble in water. 5. The support material remaining at the surface of the products was removed using a small wooden (bamboo) stick or a small wooden skewer (for instance, a small wooden stick like Japanese “Tsumayoji”) to clean up the desired GBF-holder and GBF-case carefully (the use of metal wire is not recommended as it may cause scratches on the surface). 6. Repeated the wash of GBF-holder (and GBF-case) and removal of support material several times to obtain their pure forms.

Cell Culture.


14

Page 15 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.

Mouse Lewis lung carcinoma (LLC) with enhanced green fluorescent protein (EGFP) (LLC-EGFP) and B16 melanoma cell lines were maintained in a high glucose Dulbecco’s modified Eagle’s medium.

2.

SP2/O cell line was maintained in RPMI 1640 medium supplemented with HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) and 1-thioglycerol (0.5 mM/L).

3.

MDA-MB 157 cell line was maintained in low glucose DMEM.

Cells were maintained at 37°C in a humidified 5% CO2 in their respective media supplemented with 10 % fetal bovine serum, L-glutamine and antibiotic mixture (penicillin; 69.9 ng/mL and streptomycin; 139.3 ng/mL).

Filtration, Growth, and Recovery of Cancer Cells by Means of GBF. Filtration of cancer cells (typically, LLC-EGFP) through various filters (Figure 2). 1. Filter was stacked between two plastic rings and several rubber rings and then sealed with paraffin tape to avoid leakage. 2. The filter with stacked rings were then assembled between two cylindrical shaped plastic tubes and fixed with clips for the filtration process (Figure 2a and 2b). 3. The filtration system was then placed vertically on a stand to facilitate the filtration process. 4. First, ca. 5 mL of 0.9 % saline was passed through the filtration system (to wash and fill the GBF pores) and then suspensions of LLC-EGFP cells (5 × 104 cells/mL) in 0.9 % saline (8 mL) were filtered through various filters. 5. The numbers of cells in the suspension (1 mL) before and after filtration were determined by means of a particle size analyzer.49 6. After filtration, the filters were placed upside down and their surface was observed by fluorescent microscopy. ACS Paragon Plus Environment

15


Page 16 of 54

7. In a similar manner, the capture efficiency of LLC-EGFP cells by different filters (GF, nylon mesh, polystyrene mesh, thimble and fabric (non-woven) filter) was measured as summarized in Figure 2c. The capture efficiency of filters was evaluated using the equation (4).

Capture efficiency (%) = ((initial cell number)–(cells in filtrate)/initial cell number)*100.

(4)

For comparison of filtration efficacy of cancer cells between 1.2 mm GBF and commercial filter (GF) (Figure 3). 1. A suspension of LLC-EGFP cells (1.1 × 106 cells/mL) spiked in bovine blood (5 mL) were filtered through a 1.2 mm GBF and GF (Supporting Movie S3 in Supporting Information). 2. The numbers of LLC-EGFP cells and RBCs before and after filtration were counted on a particle size analyzer, as reported in Ref. 49. 3. After filtration, LLC-EGFP cells captured on/in the filters were observed by fluorescent microscopy. 4. LLC-EGFP cells trapped on/in the GBF were incubated for 4 days. 5. After incubation for 4 days, LLC-EGFP cells grown on/in the GBF were treated with trypsin-EDTA and GBF was inserted in a GBF-holder for the cell recovery from the filter by centrifugation at 3300 rpm × 10 min and 4C (see the next section). 6. The obtained cells (pellet) were counted using a particle size analyzer/haemocytometer and then seeded at a density of 1.2 × 104 cells/well in a 24 well plate for further incubation. 7. After incubation of recovered cells for several days (2 and 3 days), the cells were imaged by a fluorescent microscope and counted by haemocytometer to check growth of cancer cells recovered from filters.

When analysis sample volume was greater than 50 L and/or expected cell count was over 2000 cells/mL, a particle size analyzer was used. When analysis sample volume was less than 50 L and/or ACS Paragon Plus Environment

16

Page 17 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


expected cell count was ca. 100~1000 cells/mL, a haemocytometer was used. Note that negligible discrepancy was observed between the two counting methods.

Comparison of filtration efficiency of 1.2 mm thick GBF with that of 0.4 mm thick GBF (Figure S10a and S10b in Supporting Information). 1. A suspension of 5 × 105 cells/mL of LLC-EGFP cells spiked in 0.9 % saline (6 mL) was filtered three times through a 1.2 mm GBF and a 0.4 mm GBF. 2. The number of cancer cells in the filtrate was counted by means of a haemocytometer. 3. The LLC-EGFP cells captured on GBF were observed by fluorescent microscopy immediately after filtration.

Evaluation of recovery efficiency of LLC-EGFP from 0.4 mm GBF (Figure S10c–10e in Supporting Information). 1. Suspension of 0.7–4.4 × 105 cells/mL of LLC-EGFP cells were spiked in pure and diluted bovine blood (3 times with 0.9 % saline). 2. The resulting suspension was then filtered through three stacked GBF assemblies (0.4 mm thickness). 3. These three GBFs were incubated separately at 37 C for three days. 4. The cell numbers before and after incubation for 3 days were determined using a particle size analyzer. 5. After incubation for 3 days, the cultured cells were recovered by trypsinization and centrifugation using a GBF-holder (3300 rpm × 10 min at 4C) (see the next section). 6. The obtained cells (pellet) were then counted using a haemocytometer and seeded at a density of 1.5 × 104 cells/well in a 24 well plate and incubated again. 7. After 4 days of incubation, the cells were counted again to confirm growth rate of recovered cells. ACS Paragon Plus Environment

17


Page 18 of 54

Use of a Specific GBF Holder (GBF-Holder) for Centrifugation-based Recovery of the Cancer Cells Trapped and Grown on/in the GBF and a GBF-Case for Cancer Cell Trap in Blood Circulation System. The centrifugation holder specific for GBF (GBF-holder) was designed to hold the GBF (and GF) during centrifugation in a commercially available centrifuge tube (50 mL) and to recover cells that were trapped and cultured in these filters (Figure S11 and Supporting Movie S4 in Supporting Information). The GBF-holder is equipped with a T-shape projection at the upper inside of the holder to permit it to be easily handled with a tweezer (Figure S11c in Supporting Information). 1. After the filtration of cancer cells through 0.4/1.2 mm GBF, the GBF was inserted into the horizontal slit of the GBF-holder (we recommend to put the upstream side of GBF to the upside) and placed in a centrifuge tube (50 mL) for centrifugation with ca. 10-20 mL media (twice at 3300 rpm × 10 min and 4C) to collect cancer cells at the bottom of the centrifuge tube (Figure S11c in Supporting Information). 2. The pellet (recovered cells from the filter) was then suspended in a suitable medium (DMEM/RPMI) and counted either by a particle size analyzer or by a haemocytometer.

A GBF-case was designed as shown in Figure S13 and Supporting Movie S5 in Supporting Information and prepared with a 3D printer to accommodate the filter during circulating (multiple) filtration (Figure 4a).

A GBF filter is horizontally placed on the nut part and then closed with the bolt

part. The GBF-case including GBF inside is then attached to the circulation system, as shown in Figure 4a, for the circulation filtration.

Capture of Cancer Cells Using GBF via the Blood Circulation System. Evaluation of capture efficiency of LLC-EGFP cells in large volume of blood samples by GBF using blood circulation system was carried out, as displayed in Figure 4. The circulation system for the ACS Paragon Plus Environment

18

Page 19 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


continuous (multiple) filtration cancer cells in a large amount of blood (~50 mL) consists of a filter case (GBF-case) to accommodate the GBF (and GF), which was constructed with a 3D printer based on a blueprint presented in Figure S13 in Supporting Information, a circulation pump and dialysis circulation tube system, which is commercially available for the dialysis of animals such as dogs and cats, was prepared as displayed in Figure 4a and Supporting Movies S5 and S6 in Supporting Information. 1. The blood circulation system was set up as shown in Figure 4a. 2. Blood samples (50 mL) containing LLC-EGFP cells (ca. 1.4 × 106 cells/mL) were placed in a bottle on ice with constant gentle agitation on the shaker during the filtration process (Figure 4a). 3. The spiked blood sample was circulated through the circulation tube system at a flow rate of 5 mL/min using a circulation (peristaltic) pump. 4. During the circulation of blood, a 0.5 mL aliquot was withdrawn from the bottles after circulation for a given time (0, 10, 20, 40, 60, 80, 100, 120 min) and diluted with 0.9 % saline/PBS (125 times) for measurement on a particle size analyzer to follow the change in the numbers of RBCs and cancer cells (Figure 4b). 5. After circulation for 2 h, the GBF was placed in a 3.5 cm dish, incubated for 2 and 4 days. 6. After incubation, the surface of the GBF was observed by fluorescent microscopy to check the growth of LLC-EGFP cells (Figure 4c) and the cells that were trapped on/in the GBF were recovered by trypsinization and centrifugation using the GBF-holder (3300 rpm × 10 min at 4C). 7. The obtained cells (pellet) were then counted using a haemocytometer and seeded in a 24 well dish at a density of 1.2 × 104 cells/well and incubated again. Cells were counted and imaged on the second and third days post seeding (Figure 4d and 4e).

The effect of the GBF and circulation system on the oxygen binding capacity of haemoglobin in the circulating blood was evaluated by UV/Vis absorbance measurement on a JASCO V-550 spectrophotometer at 25C. Bovine blood circulated in the aforementioned circulation system equipped


19


Page 20 of 54

with the GBF for a given time (2 h) was oxygenated (O2 bubbling for 90 sec) and deoxygenated (CO2 bubbling for 180 sec) to check the change in the UV/Vis absorbance spectra (Figure 4f).

Preparation of Mouse Model with Circulating Cancer Cells in the Blood, Their Growth and Recovery Using iGBF. All animal experiments were conducted in accordance with the guidelines of Tokyo University of Science (approved animal experiment number: Y17006 and S15005). A circulating cancer cells mouse model was prepared to capture circulating cancer cells by the iGBF (Figure S15 in Supporting Information). 1. A suspension of 2 × 106 LLC-EGFP cells in saline (200 μL) were injected directly into the blood of C57BL/6 mice (ca. 7 weeks old) via the tail vein using a 25 G needle (n=5) (Figure S15a in Supporting Information). 2. After circulation for 20 min, the mice were anesthetized, and their peripheral blood was collected by cardiac puncture using a 21 G needle. In these experiments, 0.5 mL~1.0 mL of blood was collected from each mouse and the same volume of blood (typically, 200 μL) was used for filtration experiments. 3. The collected blood (200 μL) was placed in an iGBF and then gently centrifuged (500 rpm × 3 min, 4C) in a 5 mL centrifuge tube to filter (trap) the cancer cells and remove (filtrate) the blood. 4. After centrifugation, the iGBF was placed in a 12 well dish and incubated for 10 days and the cancer cells that were cultured on/in the filters were recovered by trypsinization followed by two centrifugations using a 5 mL centrifuge tube (twice at 3300 rpm × 12 min at 4C). 5. The number of recovered cancer cells (pellet) was counted using a haemocytometer (Figure S15b in Supporting Information) and seeded in a 24 well plate for 24 h to check their growth and attachment, which was confirmed by a fluorescent microscopy (Figure S15c in Supporting Information). ACS Paragon Plus Environment

20

Page 21 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6. A part of the recovered cells was analyzed on a particle size analyzer (Figure S15d in Supporting Information) and the resulting cell images (Figure S15e in Supporting Information) were analyzed using the “Cancer Cell Finder (CCF) ver. 1.0” program to classify them into cancer or non-cancer cells (Figure S15f in Supporting Information),49 whose action (Macintosh version) is displayed in Supporting Movie S7 in Supporting Information (it shows the discrimination of two A549 cancer cells from two normal cells (white blood cells)).

Preparation of B16-EGFP CTC Mouse Model and Capture of CTCs from Blood, and Their Growth and Recovery. All animal experiments were conducted in accordance with the guidelines of Tokyo University of Science (approved animal experiment number: Y17006 and S15005). A B16-EGFP CTC mouse model was prepared to capture CTCs by the iGBF (Figure 5). 1. A suspension of 4 × 106 LLC-EGFP cells in 200 μL of saline per mouse (6-7 weeks old) were injected to C57BL/6 mice via the tail vein (n=6) using a 25 G needle. In control mice, 200 μL of saline per mouse was injected (n=3) (Figure 5a). 2. After 31 days, the mice were anesthetized and were cut open from the abdomen. 3. First, their peripheral blood (ca. 0.4~1 mL) was collected from heart using a heparin coated 21 G needle and then their lungs were cut out using scissors from the trachea (Figure 5b) to check for lung tumors. 4. The collected blood (350 μL) was placed in an iGBF, which was then placed in a 5 mL centrifuge tube and centrifuged (500 rpm × 3 min, 4C) to capture the CTCs. 5. After this manipulation, the iGBF was placed in a 12 well dish and incubated for 31 days with regular media change. 6. After 27 days, some loosely attached captured CTCs were cultured in a new culture dish for 4 days to observe their attachment and growth by fluorescent microscopy (Figure 5c).


21


Page 22 of 54

7. After 31 days, cancer cells grown on/in filters were recovered by trypsinization followed by centrifugation (3300 rpm × 12 min at 4C) twice.

The recovered cancer cells (pellet) were

counted using a haemocytometer (Figure 5d). 8. Some of the recovered cells were cultured for 3 days to check their attachment and growth in a 24 well dish, which was confirmed by fluorescent microscopy (Figure 5e). 9. The attached cells were then harvested by trypsinization again and analyzed using a particle size analyzer (Figure 5f and 5g). 10. Cell images obtained by particle size analyzer were subjected to the “CCF ver. 1.0” software analysis49 to judge them as cancer or non-cancer cells (Figure 5h and 5i).

■ ANTICIPATED RESULTS Design and preparation of GBF and iGBF A scheme showing the procedure for the capture, growth and collection of CTCs is presented in Figure 1. The glass-beads filters (GBF and iGBF) used to capture and culture the cancer cells were prepared as shown in Figure S1 and S2 in Supporting Information. Round-shaped glass beads with a diameter of 63−106 m (SEM images of glass beads; Figure S1g in Supporting Information) were molded in an alumina tube (Figure S1a and S1b in Supporting Information) and sintered (heated) at 700 C for 1 h to produce a GBF (Figure S1c and S1h in Supporting Information). Its pore size was adjusted by controlling the sintering time needed to trap cancer cell lines (diameter > 10 m) but not RBC (diameter < 9 m), allowing RBCs to pass through GBF, based on the threshold (around 8~9 m) between the sizes of cancer cells and RBC, as speculated in Figure S3 in Supporting Information according to our previous report.49 GBFs with a thickness of 1.2 mm and 0.4 mm and a diameter of 24 mm were prepared for in vitro experiments (Figure S1c in Supporting Information). The aforementioned conditions for the preparation of GBF (at 700 C for 1 h) were determined according to the results of the filtration experiments using polystyrene beads having 15 m and 6 m diameter as models of cancer ACS Paragon Plus Environment

22

Page 23 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cells and RBCs, respectively, described in the next section. A commercially available glass filter (diameter of 24 mm) was mechanically shaved and polished to adjust its thickness to 1.2 mm and diameter to 24 mm (without sintering) (Figure S1j and S1k in Supporting Information). The density and porosity of the GBF was 1.7 g/cm3 and 30.1 %, respectively, whereas the density and porosity of the GF was 1.3 g/cm3 and 40.2 %, respectively. We also designed and prepared a small integrated glass-beads filters (iGBF) with a diameter of ca. 9.6 mm and a thickness of ca. 2 mm (as for glass-beads filter part), as displayed in Figure S1d-1f and S1i in Supporting Information, to use in conjunction with smaller amounts of blood samples from small animals such as mice. It should be noted that the size of the entire filter and its pore can be controlled by choosing appropriate glass beads and the conditions used for their preparation.

Filtration efficiency measurement of GBF using polystyrene beads The filtration properties of the 1.2 mm thick GBF were initially tested using polystyrene beads with diameters of 15 m and 6 m as models of cancer cells and RBCs, respectively. Polystyrene beads were filtered in an apparatus shown in Figure S4a in Supporting Information and changes in the concentrations of polystyrene beads before and after filtration were assessed by UV/Vis absorption spectra (calibration curves are shown in Figure S4b in Supporting Information). As shown in Figure S5a in Supporting Information, 15 m diameter polystyrene beads were efficiently trapped by the GBF (up to ca. 94 %), while only ca. 17 % of the 6 m diameter polystyrene beads were trapped, suggesting that the GBF traps 15 m polystyrene beads more efficiently than 6 m polystyrene beads. As shown in Figure S5b in Supporting Information, the clear polystyrene dispersion of 15 m beads after filtration through GBF implies that the 15 m polystyrene beads were efficiently trapped compared to the 6 m polystyrene beads. In addition, the movement of 15 m polystyrene beads through the GBF and GF was observed by high-speed camera, as displayed in Supporting Movie S1 and S2 in Supporting Information. ACS Paragon Plus Environment

23


Page 24 of 54

Prior to the aforementioned experiments, we prepared several types of GBFs from glass beads that have different diameter and size distribution by sintering at 700 oC. As summarized in Table S1 in Supporting Information, glass beads in Entries 1 and 2 have narrow size distribution at around 70  5 and 100  5 m, respectively, and those in Entries 3 and 4 have broad size distribution at around 63~106 and 90~125 m, respectively (glass beads were purchased from Union Co. Ltd., Japan). The measurement of pore sizes of GBFs prepared by sintering at 700 oC for 1 h, as displayed in Figure S6a in Supporting Information (schematic presentation) and Figure S6b and S6c in Supporting Information (real images of the surface of GBF) revealed that glass beads in Entries 2 and 3 have almost same pore size of ca. 65 m (see Figure S6b in Supporting Information) and those in Entries 1 and 4 have smaller and greater pore size (40 m in Entry 1 and ca. 80 m in Entry 4), respectively (see Figure S6c for Entry 4), as summarized in Table S1 in Supporting Information. Figure S7 in Supporting Information shows the results of filtration of polystyrene beads with diameters of 15 m (model of CTCs) and 6 m (model of RBCs) through GBFs prepared in Table S1, implying that GBFs prepared in Entries 2 and 3 have almost same filtration properties and the capture efficiency of 15 m polystyrene through the GBF in Entry 4 is lower (passing ratio is higher) than those of Entries 2 and 3. It was also found that the passing ratio of 6 m polystyrene beads through GBF obtained by sintering for 2 h at 700 oC (by sintering 63~106 m glass beads at 700 oC for 1 h) was reduced to ca. 60% (Entry 5 of Table S1 and Figure S6d and S7 in Supporting Information). Because 63~106 m glass beads used in Entry 3 (Table S1 in Supporting Information) are cheaper than those in Entry 2, we decided to use 63~106 m glass beads for GBF preparation (Entry 3 of Table S1 in Supporting Information) for further experiments. The results of the detailed analysis of the movement of polystyrene beads in the GBF as well as the effects of preparation conditions of GBF on its filter structure and filtration property will be reported elsewhere.

Capture of cancer cells by GBF ACS Paragon Plus Environment

24

Page 25 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The aforementioned results prompted us to examine the filtration of Lewis lung carcinoma cells that overexpress green fluorescent protein (LLC-EGFP cells), thus permitting easy detection on fluorescent microcopy and observation during the experiments. Quite recently, we reported on the marker free detection of cancer cells and clusters in vitro by using a particle size analyzer (Jasco-Occhio particle size analyzer, IF nano-200), which detects size parameters of live cells such as inner diameter and shape parameters such as circularity and roundness.49 We reported that cancer and non-cancer cells can be discriminated based on the difference in their inner diameter. Furthermore, the detection of the cancer cells spiked in mouse/human blood using a particle size analyzer was also reported. Therefore, the filtration efficiency of LLC-EGFP cells alone and those cells spiked in bovine blood by the GBF were easily assessed using a fluorescent microscopy and particle size analyzer. For the filtrations of cancer cells, a filter such as GBF is assembled between two plastic rings and several rubber rings using two cylindrical shaped plastic tubes (Figure 2a and 2b) and placed vertical on a stand to facilitate the filtration process. The assembly is tightly sealed with a paraffin tape to avoid the leakage of samples (Figure 2b). To evaluate the efficiency of capturing cancer cells of the GBF and various commercially available filters, LLC-EGFP cells were spiked in saline and then passed through filters placed in the assembly shown in Figure 2b. Figure 2c implies that capture efficiency of LLC-EGFP cells by GBF, GF and thimble filter are 91 %, 99 %, and 98 %, respectively, and that nylon mesh, polystyrene mesh and fabric filters had capture efficiencies of 33-41%, 43 % and 71 %, respectively. The LLC-EGFP cells trapped by various filters were immediately observed by fluorescent microscopy, as shown in Figure S8 in Supporting Information. Single sheet filters such as nylon mesh, polystyrene mesh and fabric filter do not support cancer cell growth well and hence cannot be used for repeated filtration. It should be noted that, although the thimble filter (Figure S9 in Supporting Information) trapped LLC-EGFP cells efficiently, but it was not used for further experiments, because it is made of cellulose and it was assumed to be unsuitable for use in repeated filtration described later. Because GF also has efficient capture efficiency and is made of glass like GBF, GF was used for further experiments for comparison with GBF. ACS Paragon Plus Environment

25


(a)

GBF/GF

(b) Filter

Plastic ring Tape Filter Rubber rings

Filtrate Cell number count

(c) Capture efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 54

120 100 80 60 40 20 0

GBF

CF GF

1010 μm

7 7μm 1010 μm Thimble Fabric filter filter Nylon mesh Polystyrene mesh

Figure 2. Filter assembly and capture efficiency of the GBF. (a) Modules of filtering system for capturing cancer cells. (b) Photo of single filtration system. (c) Capture efficiency (%) of various filters to trap LLC-EGFP cells with respect to the initial numbers of LLC-EGFP cells (5 × 104 cells/mL of saline).

Capture and growth of cancer cells spiked in blood by a 1.2 mm thick GBF


26

Page 27 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We next evaluated the ability of the GBF to capture cancer cells spiked in blood and to grow cancer cells on/in filters, as well as assessment of any adverse effects on the proliferation characteristics of the captured cancer cells. LLC-EGFP cells spiked in bovine blood (containing 10~20 U/mL heparin) were captured by single filtration (Figure 2b and Supporting Movie S3 in Supporting Information) through a 1.2 mm GBF and GF. The numbers of cancer cells and RBCs before and after filtration were counted on a particle size analyzer. As shown in Figure 3a and 3b, the number of cancer cells after filtration through the GBF decreased considerably (capture efficiency: 46 %, total captured cell number: ca. 2.5 × 106/5 mL of filtration sample), which was greater than that of the GF (capture efficiency: 19 %, total captured cell number: ca. 1.1 × 106/5 mL of filtration sample).50 The capture efficiency of GBF for spiked sample (capture efficiency: 46 %) was lower than that for 15 µm diameter polystyrene beads (capture efficiency: ca. 94 %) (Figure S5 in Supporting Information) and LLC cells in saline (capture efficiency: ca. 91 %) (Figure 2c). Our observation suggests that LLC cells are more flexible (softer) than polystyrene beads and hence go through the pores of GBF, resulting in its lower capture efficiency than that of 15 µm polystyrene beads. A small change in the number of RBCs after filtration through the GBF was observed, implying that most of the RBCs pass through the GBF. LLF-EGFP cells that were captured in the GBF and GF were incubated for 4 days and their growth was observed by fluorescent microscopy at the second and fourth day post filtration, as shown in Figure 3c, indicating that LLC-EGFP cells were alive and can be cultured on the GBF. After incubation for 4 days, the grown LLC-EGFP cells were collected by trypsinization and again seeded in new culture dishes at an equal seeding density and their proliferation was assessed for 2-3 days with respect to control LLC-EGFP cells (cells not used for filtration and grown in a culture dish) for comparison. As shown in Figure 3d and 3e, the difference in proliferation (cell number) was negligibly observed between the LLC-EGFP cells recovered from the GBF and control cells, implying the GBFs caused negligible damage to the captured cancer cells. On the other hand, the reduced proliferation of LLC-EGFP cells recovered from GF suggests that the proliferative ACS Paragon Plus Environment

27


Page 28 of 54

characteristics of captured cells are somewhat damaged by the GF (Figure 3f). Microscopic images shown in Figure 3e and 3g suggest that the cells recovered from filters retain their adherent characteristics even after growth on the filters.


28

Page 29 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Comparison of capture and growth of cancer cells between 1.2 mm GBF and GF. Change in the numbers of RBC (initially ca. 6.6 × 109 cells/mL) (red bars) and LLC cells (ca. 1.1 × 106 cells/mL ACS Paragon Plus Environment

29


Page 30 of 54

bovine blood; filtration volume = 5 mL) (black bars) in bovine blood containing LLC cells before and after filtration through the GBF (a) and the GF (b), as evaluated by a particle size analyzer. After filtration the cancer cells that were captured on/in the filters were grown for 4 days; (c) microscopic images (GFP and an overlay of the bright field and GFP) of captured cells immediately after filtration and incubation for 2 days and 4 days. Grown LLC-EGFP cells were recovered by trypsinization and seeded on culture dishes at an equal seeding density and assessed for proliferation with respect to control (cells not captured and grown on filters); (d) proliferation of LLC-EGFP cells recovered from GBF, (e) microscopic images of recovered cells from GBF incubated for 2 days and 4 days, (f) proliferation of LLC-EGFP cells recovered from GF and (g) microscopic images of recovered cells from GF incubated for 2 days and 4 days. Scale bar = 50 m.

Recovery of the captured cancer cells from a 0.4 mm GBF After the successful growth of the cancer cells captured on GBF, we next attempted at the recovery of the cancer cells from the filters. Since it was assumed that the recovery of the cancer cells from a 1.2 mm filter may not be so efficient (because the cells are embedded inside the thick filter), a thinner GBF (0.4 mm thick) was prepared. Firstly, the efficiency of capture of LLC-EGFP cells by a single 0.4 mm GBF was compared with that of a 1.2 mm GBF. LLC-EGFP cells in saline were filtered through a single filtration assembly (Figure 2b) three times and the number of captured cancer cells were counted. As shown in Figure S10a in Supporting Information, the capture efficiency of both GBFs (1.2 mm and 0.4 mm) increased with increasing filtration times. The capture efficiency for cell samples that were filtered three times through the 0.4 mm GBF was almost same as that after single filtration through the 1.2 mm GBF. Microscopic images showing LLC-EFGP cells on both filters after three times filtrations confirmed that cancer cells were captured during the filtration (Figure S10b in Supporting Information).


30

Page 31 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Next, LLC-EGFP cells spiked in non-diluted bovine blood and diluted bovine blood (three times with saline) were filtered through the stacked three 0.4 mm GBF (single filtration) (Figure S10c in Supporting Information). Negligible pressure was required for the filtration of diluted blood samples, while a slightly stronger air pressure was required for the filtration of non-diluted samples.51 After filtration, the cells that were captured on/in the filters were incubated in DMEM medium for 3 days. Figure S10d in Supporting Information shows microscopic images of the surface of three 0.4 mm GBF (top, middle and bottom) immediately after filtration and after incubation for 3 days, confirming that the LLC-EGFP cells were, in fact, captured and were able to grow in/on top, middle, and bottom filters. More importantly, these results strongly imply that cancer cells are trapped not only on the surface of the GBF but also inside the GBF. After 3 days, the grown LLC-EGFP cells were detached from filters by the treatment with trypsin and recovered by centrifugation. In this manipulation, the filter was gently placed in a GBF-holder (Figure S11 in Supporting Information), which was designed and constructed with a 3D printer in this work for culturing trapped cancer cells (in 6 well plates) and for their convenient recovery from filters with minimal damage. After filtration, the GBF-holder containing the GBF was then placed in a centrifuge tube (50 mL) and centrifuged (3300 rpm × 10 min, 4C) to collect the grown LLC-EGFP cells at the bottom of a centrifuge tube (for these manipulations, see Supporting Movie S4 in Supporting Information). The recovery efficiency of cancer cells from the GBF after incubation for 3 days with respect to the initial number of cancer cells that had been spiked in blood was higher in the diluted blood than that for the non-diluted blood sample (Figure S10c in Supporting Information), suggesting that the relatively high air pressure used for the filtration of cancer cells spiked in non-diluted blood might have caused considerable damage to the cells. The number of captured cancer cells was found to be the highest in the top filter, followed by the middle filter and then bottom GBF in the diluted blood sample (Figure S10c in Supporting Information). The proliferative properties of the recovered cancer cells were also assessed after incubation for 4 days. As shown in Figure S10e in Supporting Information, negligible ACS Paragon Plus Environment

31


Page 32 of 54

difference in cell number was observed in the case of the diluted blood sample after recovery from the three filters, suggesting that the cells had been negligibly damaged during filtration and recovery under these experimental conditions. Furthermore, the capture and recovery efficiency of the 0.4 mm GBF and GF were tested by a single filtration of LLC-EGFP cells in saline through three stacked 0.4 mm GBF and GF. As shown in Figure S12a in Supporting Information, the capture efficiency values for the LLC-EGFP cells by the GBF and GF were 80 % and 85 %, respectively. The efficiency of recovery of LLC-EGFP cells from the GBF and GF after 3 days of incubation was 85 % and 81 %, respectively, suggesting that the efficiency of capture of GBF is slightly lower than that for GF, while the growth and recovery of LLC-EGFP cells captured on the GBF is better than that on GF (Figure S12b in Supporting Information). Microscopic images of the filters (top, middle and bottom) confirmed the capture and growth of LLC-EGFP cells, immediately after filtration and after incubation for 3 days, respectively, for GBF and GF (Figure S12c and S12d in Supporting Information).

Multiple (repeated) filtration of cancer cells spiked in blood by GBF in a circulation system The aforementioned results prompted us to test whether very small number of cancer cells in blood could be captured from large amount of blood samples (typically, from blood samples of human patients or cancer animals) by the GBF in a circulation filtration system (repeated filtration process). In this work, a filtration circulation system was assembled with a filter case (GBF-case, not GBF-holder described above) to accommodate a GBF (Figure S13 and Supporting Movie S5 in Supporting Information), a circulation (peristaltic) pump (to control the circulation speed) and a commercially available circulation tube system for dialysis (Figure 4a). LLC-EGFP cells spiked in bovine blood (placed in glass bottles on ice) were circulated at a flow rate of 5 mL/min for 2 h through the circulation system equipped with a 1.2 mm GBF (see Supporting Movie S6 in Supporting Information, in which blood samples are circulated with the negligible formation of blood clots in this system even at 10~25 mL/min). The spiked samples were constantly and gently agitated on ice to ensure the proper mixing ACS Paragon Plus Environment

32

Page 33 of 54

of cancer cells in the blood. Aliquots of blood were removed during the circulation at specified times (0, 10, 20, 40, 60, 80, 100, and 120 min) and were analyzed on a particle size analyzer to follow changes in the numbers of cancer cells and RBCs.

No filter

GBF-case

(b)

(c)

7 6

(c) RBC (×109/mL)

Circulation system

2

LLC cells spiked in blood

1.6

5 4

1.2

3

0.8

2

0.4

1 0

4 days incubation

2 days incubation GFP

GBF in GBF-case

Bright field and GFP

(a)

LLC cells (×106/mL)

0 -100 0 100 200 300 400 500 600 700

Shaker

Circulation pump

Total circulated volume (mL)

GBF

15 10 5 0 Initial

248days h

372 days h

1.2

2

1 CO

1.6 1.4

Control cells Absorbance

20

Captured cells from GBF

Control

3 days incubation

LLC cells (×104)

25

2 days incubation

30

(f)

0.8

+

(e)

(d)

Absorbance at 760 nm

1.2 + 2 O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6 0.4

0.5

1

1.5

2

2.5

3

3.5

1 0.8 0.6 0.4 400 500 600 700 800 900 1000 Wavelength (nm)

Figure 4. Capture of cancer cells spiked in blood by the circulation filtration method. (a) Photo of the circulation system encompassing; GBF-case (inset) to accommodate GBF, circulation pump, dialysis circulation tubes. Cancer cell spiked sample in glass bottles were placed on ice with constant gentle agitation during the circulated filtration to ensure uniform distribution of cancer cells in the suspension. (b) LLC-EGFP cells (1.4 × 106 LLC cells/mL) spiked in bovine blood (50 mL) were passed through the circulation system equipped with a GBF at a flow rate of 5 mL/min. Aliquot of blood samples were withdrawn at 0, 10, 20, 40, 60, 80, 100, and 120 min and analyzed on a particle size analyzer to follow changes in numbers of cancer and blood cells. A considerable reduction in the number of LLC-EGFP


33


Page 34 of 54

cells (blue circles) (capture by GBF) with negligible change in RBCs (red circles) were observed. After filtration, the cancer cells that were captured on the GBF were incubated for 2 and 4 days. (c) Microscopic images (GFP and an overlay of the bright field and GFP) of captured cells after filtration and incubation for 2 and 3 days, scale bar = 50 m. Grown LLC-EGFP cells were recovered by trypsinization and seeded into culture dishes at an equal seeding density and assessed for proliferation with respect to the control cell sample (cells not captured and grown on filters); (d) Proliferation of LLC-EGFP cells recovered from the GBF and incubated for 2 and 3 days, (e) Fluorescence microscopic images of the cells recovered from the GBF and incubated for 2 and 3 days, scale bar = 50 m. (f) Effect of GBF and circulation system on the oxygen binding capacity of haemoglobin of circulated RBCs; absorbance spectra of blood immediately after filtration (red line), oxygenated blood O2 bubbling for 90 sec; dashed blue line) and deoxygenated circulated blood (CO2 bubbling for 180 sec; green line) (inset; change in absorbance at 760 nm of haemoglobin in the circulated red blood cells immediately after filtration, after oxygenation and deoxygenation).

As shown in Figure 4b, a considerable reduction in the number of LLC-EGFP cells was observed with increasing circulation time and capture efficiency of the GBF after the circulation for 2 h was ca. 71 %; ca. 50 × 106 LLC-EGFP cells were trapped on the GBF from the initial 70 × 106 cells. In addition, only a small decrease in the number of RBCs was observed during the circulation, suggesting that the LLC-EGFP cells had been selectively captured by the GBF. After circulation, the GBF was incubated for 4 days to allow the captured cancer cells to grow and microscopic images shown in Figure 4c suggest that these cells were successfully captured and grown. Furthermore, the grown LLC-EGFP cells were recovered for proliferation assay. As shown in Figure 4d, the difference in the increase in the cell numbers between control cells (LLC-EGFP cells that were not subjected to the circulation system and grown on GBF) and cancer cells recovered from GBF at second and third day after recovery from GBF were negligible, suggesting that the captured cancer cells during circulation


34

Page 35 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and growth on/in the GBF suffered negligible damage. Microscopic images shown in Figure 4e imply that the recovered LLC-EGFP cells retained their adherent characteristics. For comparison, LLC-EGFP cells spiked in bovine blood were circulated in the circulation system without GBF at the same flow rate (5 mL/min) (Figure S14 in Supporting Information). Blood samples were analyzed at 0, 10, 20, 40, 60, 80, 100, and 120 min on the particle size analyzer to monitor change in the number of cancer and RBCs. A negligible decrease in the number of LLC-EGFP and RBCs was observed (Figure S14b in Supporting Information), suggesting the circulation system without GBF had a negligible effect on the component of the blood samples containing cancer cells. Next, the effect of the circulation process on the oxygen (O2) binding properties of haemoglobin in RBCs was examined. UV/Vis absorbance spectra of blood samples were obtained immediately after circulation for 2 h with the GBF, as shown in Figure 4f. The absorbance at 760 nm (corresponding to haemoglobin) was measured for the same blood sample after oxygenation and deoxygenation steps. As shown in the inset of Figure 4f, the absorbance of haemoglobin at 760 nm was low immediately after filtration (because the samples were exposed to O2 during circulation in our experiments) and the absorbance was increased by CO2 bubbling in a reversible manner, suggesting that the circulation of RBCs through GBF negligibly affected the O2 binding property of haemoglobin.

Capture, growth and recovery of circulating cancer cells from a circulating cancer cells mouse model by integrated GBF (iGBF) To capture cancer cells from in vivo animal blood samples using the GBF, we injected LLC-EGFP cells to C57 mice (2 × 106 cells/mouse) and collected their blood after 20 min (Figure S15a in Supporting Information). Blood (not diluted) samples (ca. 0.3~0.6 mL) were then filtered through 1.2 mm GBF and the cells that were trapped on/in the iGBF were cultured in a 3.5 cm dish. After incubation for 3 days, the grown cancer cells were recovered by trypsinization followed by centrifugation and then manually counted. As shown in Figure S15b in Supporting Information, many GFP-positive cells (44 GFP-positive cells/10000 lung cells) were observed in lung samples of mice that ACS Paragon Plus Environment

35


Page 36 of 54

had been injected with LLC-EGFP cells. After filtering blood samples obtained from LLC-EGFP injected mice, the cancer cells trapped on/in the GBF were incubated for 3 days and observed by fluorescent microscopy. We observed only few scattered GFP-positive cells on the filters (Figure S15c in Supporting Information). Although the capture + recovery efficiency of the circulating cancer cells from GBF was 0.013% with respect to the number of cells that were originally injected (Figure S15d in Supporting Information), the capture of the LLC-EGFP cells that had been injected into the mouse and their reculture was successful. As shown in Figure S15e in Supporting Information, the recovered cancer cells retained their adherent characteristics even after incubation for 7 days. In order to treat a small amount (~ 0.1 mL) of blood, we designed a small GBF (referred to as an integrated GBF, iGBF) with a diameter of 9.6 mm sandwiched between the glass tubes (Figure S1d-1f in Supporting Information). First, we measured the capture efficiency of iGBF for B16-EGFP cancer cells spiked in mouse blood (from C57 mice), as shown in Figure S16a in Supporting Information. Capture efficiency of iGBF was ca. 72 % (4.7 × 105 captured cells with respect to 6.5 × 105 initial cell number). After filtration, the captured cells were grown in iGBF, recovered (by trypsinization and centrifugation), and observed on fluorescent microscopy. As shown in the Figure S16b in Supporting Information, the recovered cells were GFP-positive, implying the recovery of B16-EGFP cells. The recovered B16-EGFP cells were assessed in a particle size analyzer, which confirmed the inner diameter of majority cells was greater than 10 m (Figure S16c in Supporting Information), as reported earlier by us.49 Typical manipulation for capture and growth of circulating cancer cells by means of the iGBF is presented in Figure S17a in Supporting Information, in which 0.15 mL blood obtained from a circulating cancer cells mouse model (injected with LLC-EGFP cells) was poured into iGBF and centrifuged in a 5 mL centrifuge tube. After this step, the cancer cells trapped on/in the iGBF were cultured for 10 days, recovered by trypsinization, and collected by centrifuge again. The number of LLC-EGFP cells recovered after a period of 10 days incubation was ca. 5.2 × 104 cells, which corresponds to ca. 34 % with respect to the numbers of LLC-EGFP cells originally injected into the ACS Paragon Plus Environment

36

Page 37 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mouse (Figure S17b in Supporting Information). These numbers are higher than the estimated number of CTCs in a cancer patient, suggesting that iGBF may be capable of efficiently capturing CTCs (and further growth) even from a small amount of blood. After recovering the captured cancer cells from iGBF, the cells were incubated for 24 h in a culture dish and observed by fluorescent microscopy. As shown in Figure S17c in Supporting Information, the recovered cells were GFP-positive, while GFP expressing cells were negligibly observed in control samples (blood from mice injected with only saline), suggesting that the LLC-EGFP cells were successfully captured and recovered by the iGBF. In addition, adverse effects on the adherent characteristics of the recovered LLC-EGFP cells were observed to be negligible. Next, the cells recovered from iGBF were analyzed using a particle size analyzer, as we reported previously.49 A scatterplot of the recovered cells with respect to inner diameter (μm) (x-axis) and circularity (%) (y-axis) (Figure S17d in Supporting Information), in which each dot in a scatterplot presents a cell/debris/cluster, suggests that the majority of cancer cells have an inner diameter of > 10 μm. We recently developed a PC software program “Cancer Cell Finder (CCF) ver. 1.0” to differentiate between cancer cells and normal cells based on our observation that cancer cells have a rougher surface than normal cells, as imaged by a particle size analyzer,49 and the sum of stationary points on darkness (brightness) curves on the cell surface through two lines (horizontal and vertical lines) are > 10, while the number in normal cells is < 10 (Supporting Movie S7 in Supporting Information displays the function of “CCF ver. 1.0” (Macintosh version) for the discrimination of two human cancer cells (A549 cells) from two normal cells (human white blood cells). Several cells observed with inner diameters greater > 10 μm obtained from particle size analyzer were subjected to analysis by the “CCF” to characterize whether they were cancer or non-cancer cells (Figure S17e and S17f in Supporting Information). As shown in Figure S17f, nine cells recovered from the iGBF have total stationary points > 10, thus judging them as cancer cells. These data suggest that the iGBF is a useful tool for capturing circulating cancer cells, growing, and recovering them efficiently.


37


Page 38 of 54

Capture, growth and recovery of circulating melanoma B16 tumor cells from in vivo tumor mouse model using an iGBF The aforementioned positive results encouraged us to attempt to capture CTCs from in vivo mouse model. For the preparation of CTC mouse model, C57 mice were injected with a B16-EGFP melanoma cell line (4 × 106 cells/mouse) and kept for 13 days (Figure 5a). As shown in Figure 5b, the lungs of mice these mice had black B16 melanoma colonies (because melanoma cells express melanin) compared to control mice that were injected with saline, confirming the successful formation of B16 tumor metastasis. Blood samples (0.35 mL) harvested from these B16 tumor mice were filtered through the iGBF to capture the B16 CTCs (Figure 5a). For comparison, control blood (0.35 mL) from a healthy mouse that had been injected with only saline was also filtered through the iGBF. The captured cells on/in the iGBF were incubated for 31 days with regular media changes to allow them to grow. During media change, the non-adherent blood cells such as white blood cells (WBCs) and RBCs are removed, as such their negligible interference is observed during CTC culture and detection. During the culture process, CTCs that were weakly bound to the iGBF and released from the surface of glass filters were collected separately and incubated in a culture dish for 4 days. As shown in Figure 5c, the attached GFP-positive cells were identified as B16-type cells. After incubation for 31 days, the captured CTCs were detached with trypsin and collected by centrifugation, which was determined to be 10.4 × 104 cells on average, as counted by a hemocytometer (Figure 5d). The recovered CTCs were plated on culture plates and visualized by fluorescent microscopy immediately after their recovery (Figure 5e). The recovered CTCs were then cultured for 3 days. The presence of GFP-positive cells in tumor bearing samples indicated EGFP expressing B16 melanoma cells (Figure 5e). The extended and attached morphology of GFP-positive B16 melanoma cells indicated that the recovered cancer cells were alive and capture by the iGBF had negligible effects on their adherent characteristics (Figure 5f). In addition, the recovered cells were analyzed on the particle size analyzer. Scatterplot of the analyzed samples indicated that majority of the recovered cells had an inner diameter of > 10 μm (Figure 5g). Analysis of several cells in Figure 5g by the “CCF ver. 1.0” indicated that the ACS Paragon Plus Environment

38

Page 39 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analyzed cells had total stationary points of > 10, strongly suggesting that these are cancer cells (Figure 5h and 5i) rather than WBCs that have similar size as that of cancer cells but have smoother surface49 than that of cancer cells.52

Figure 5. Capture, growth and recovery of melanoma B16-EGFP CTCs. (a) Scheme showing the preparation of the melanoma B16-EGFP metastasis mouse model, the capture of B16 CTCs by the iGBF, their growth, recovery and confirmation by particle size analyzer and the “CCF” software. For preparing a CTC model, mice were injected with 4 × 106 B16-EGFP cells/ mouse via the tail vein and after 13 days CTCs in the blood were captured by GBF. (b) Photos of lungs harvested at 31 days after injecting B16 melanoma cells; control mice that were injected with saline had clear lungs compared to ACS Paragon Plus Environment

39


Page 40 of 54

the lungs of B16 tumor mice model with black B16 colonies. (c) Microscopic images of B16-EGFP cells after 4 days recovered from filters at 4x magnification (scale bar = 500 m) and 20x magnification (scale bar = 50 m) (Biorevo, BZ-9000, Keyence microscope). (d) Number of recovered B16 CTCs from filters after 31 days of in vitro incubation, (e) microscopic images of recovered B16 CTCs, and (f) microscopic images of recovered B16 CTCs after 3 days of incubation, scale bar = 50 m. (g) Scatterplot of B16 CTCs (recovered from filters) analyzed on a particle size analyzer. (h) Single cell images of recovered cells from filters imaged by a particle size analyzer with their respective inner diameter (m). (i) Images from (h) were analyzed by the “CCF” software and judged as cancer cells using stationary point method. Cancer detection threshold of the “CCF” should be greater than 10 stationary point (dashed line).

■ CONCLUSION Easy and convenient methods for the capture of cancer cells in blood by a GBF and an iGBF, their culture and recovery on/in the same glass filter are reported. The developed GBF has a unique porous architecture constructed with a suitable porosity that permits the selective capture of CTCs, which generally have a greater size than that of RBCs. Filtration experiments confirmed that the efficiency of GBF to capture LLC-EGFP cells spiked in saline was above 80 % (corresponds to 4.6 × 104 cells/mL from 5.0 × 104 cells/mL) and that in bovine blood was ca. 40~50 % (corresponds to 0.5 × 106 cells/mL from 1.1 × 106 cells/mL). The efficiency of GBF to capture cancer cells from a large amount of blood (50 mL) by repeated filtration through the circulation system equipped with the GBF approached 70 % (corresponds to 1.0 × 106 cells/mL from 1.4 × 106 cells/mL) after circulation for 2 h with very low capture of RBCs. Effects on the growth and proliferative characteristics of the captured cancer cells and the O2 binding capacity of RBCs were observed to be negligible. Furthermore, the capture of B16 CTCs from the blood of a melanoma B16 tumor mouse model, their growth and recovery were also successfully carried out by means of the iGBF.


40

Page 41 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We conclude that advantages of GBF over other CTC capture techniques includes: i) the starting material and the glass beads used in these methods are inexpensive and the cost of one GBF and iGBF is less than 1 USD, ii) the preparation of the GBF is quite simple, easy and applicable to a wide range of shapes and sizes for various purposes (such as the iGBF) to treat ca. 100 μL~50 mL (or greater volumes) of blood samples (diluted and non-diluted blood can be filtered). On the other hand, the preparation of various forms from commercially available glass filters is difficult. iii) GBF and iGBF are suitable for the single and multiple (repeated or circulated) filtration of experimental and blood samples containing cancer cells, iv) the products, GBF and iGBF, are stable under experimental conditions used in this work, v) GBF negligibly induces blood clots, as long as we use, vi) the cancer cells trapped on/in GBF or iGBF can be cultured in media for several days~one month, vii) the cultured cancer cells can be recollected from the GBF using the commercially available 50 mL centrifuge tube with the specific GBF-holder, which is also readily available (3D printer), or from iGBF itself by using a 5.0 mL centrifuge tube, and viii) GBF and iGBF can be used in a repeated manner, if necessary.

The

methods using GBF and iGBF for the capture and culture of CTCs in a combination with “dry” methods for the detection of specific cells such as PC software “CCF” would be useful for characterization of CTCs, which will assist in significant prognosis information and metastatic cancer theranostics.

■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +81-4-7121-3670. Fax: +81-4-7121-3670. ORCID Babita Shashni: 0000-0002-1025-9281 Shin Aoki: 0000-0002-4287-6487 Authors Contributions


41


B.S. and S.A. (Aoki) designed, performed the experiments and wrote the manuscript. and A.Y. designed and prepared GBF and iGBF.

Page 42 of 54

H.M., R.S., T.H.,

K.N. and H.T. prepared GBF-holder and GBF-case.

S.A. (Ariyasu) and K.A. carried out the preliminary experiments on capture of cancer cells.

N.A.

developed “CCF ver. 1.0” software.

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 15H04129 for A.Y. and Nos. 24640156, 15K00408, 16K10396, and 17K08225 for S.A.) and “Academic Frontiers” project for private universities: matching funds from MEXT, and the Tokyo University of Science fund for strategic research areas. We wish to acknowledge Prof. Ryo Abe, Prof. Masato Kubo, and Prof. Hiroyasu Esumi (Research Institute for Biomedical Science, Tokyo University of Science), Prof. Masanori Hayase (Faculty of Science and Technology, Tokyo University of Science), Dr. Masahiro Motosuke (Faculty of Engineering, Tokyo University of Science), Dr. Ken-ichiro Iwasaki and Dr. Takayaski Nakanishi (Faculty of Industrial Science and Technology) and Dr. Tomohiro Osaki and Dr. Norhiko Itoh (Faculty of Agriculture, Tottori University) for their helpful discussions. We are also thankful to Mr. Yoichiro Ishikawa (Clever Co., Ltd.) and Mr. Toshiya Saito (Tapyrus Co., Ltd.) for their helpful suggestions and providing nylon mesh and fabric filters, respectively, and to Dr. Masaki Komori, Mr. Shirokazu Hirabayashi, and Mr. Kazuaki Kosaka (Leaf International, Inc., Tokyo, Japan) for helpful suggestions.

■ASSOCIATED CONTENT


42

Page 43 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Supporting Information The following files are available free of charge on the ACS Publications website at DOI: 10/1021.acsboimaterials.xxxxxxxx. Figure S1. Preparation of a glass-beads filter and an integrated glass-beads filter I. Figure S2. Preparation of a glass-bead filter and an integrated glass-beads filter II.

Figure S3. The size

distribution by volume of blood cells and cancer cells (SP2/O) analyzed on particle size analyzer. Figure S4. Assembly for the filtration of polystyrene beads and calibration curves. Figure S5. Filtration of polystyrene beads as model particles of cancer cells and RBCs. Figure S6. Pore size estimation of GBFs. Figure S7. Filtration of polystyrene beads through GBF made of glass beads with various diameter. Figure S8. Microscopic bright field, GFP and overlay images of LLC-EGFP cells captured on various filters. Figure S9. Picture of thimble filter. Figure S10. Recovery of captured cancer cells from 0.4 mm GBF. Figure S11. Centrifuge holder (GBF-holder) to hold glass filters (GBF and GF) for recovering cells cultured on/in the GF and GBF. Figure S12. Comparison of capture efficiency between a 0.4 mm GBF and the GF (three stacked filters). Figure S13. A picture and a blueprint of a case to hold the GBF used in the blood circulation system to trap cancer cells. Figure S14. Comparison of circulation filtration with and without GBF. Figure S15. Capture of circulating cancer cells by 1.2 mm GBF. Figure S16. Capture of B16-EGFP cancer cells spiked in mouse blood by iGBF. Figure S17. Capture, growth and recovery of circulating LLC-EGFP cells using the iGBF. Table S1. Glass beads with different size distribution used for the preparation of GBF and the pore size of the formed GBF by sintering at 700 oC for 1 h/2 h. Supporting Movie S1. Movement of 15 m polystyrene beads through GBF. through GF.

Supporting Movie S2. Movement of 15 m polystyrene beads

Supporting Movie S3. Single filtration of bovine blood.

Supporting Movie S4.

Demonstration on the use of GBF-holder for culture and recollection of cancer cells. Supporting Movie S5. Demonstration on the use of GBF-case for repeated (circulation) filtration system.


43


Page 44 of 54

Supporting Movie S6. Repeated (circulated) filtration of 50 mL of blood samples.

Supporting

Movie S7. A movie that displays the function of PC software “CCF ver. 1.0” (on Macintosh).

■ REFERENCES (1)

Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W. M. M.; Hayes, D. F. Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. New Engl. J. Med. 2004, 35, 781-791, DOI: 10.1056/NEJMoa040766.

(2)

Spano, D.; Heck, C.; De Antonellis, P; Christofori, G; Zollo, M. Molecular Networks that Regulate

Cancer

Metastasis.

Cancer

Biol.

2012,

22,

234-249,

DOI:

10.1016/j.semcancer.2012.03.006. (3)

Mehlen, P; Puisieux, A. Metastasis: a Question of Life or Death. Nature Reviews Cancer 2015, 6, 449-458, DOI: 10.1038/nrc1886.

(4) MoCellin, S.; Hoon, D.; Ambrosi, A.; Nitti, D.; Rossi, C. R. The Prognostic Value of Circulating Tumor Cells in Patients with Melanoma. Clin. Cancer Res. 2006, 12, 4605-4613, DOI: 10.1158/1078-0432.CCR-06-0823. (5) Paoletti, C; Li, Y; Muñiz, M. C; Kidwell, K. M; Aung, K; Thomas, D. G.; Brown, M. E; Abramson, V. G.; Irvin Jr, W. J.; Lin, N. U.; Liu, M. C.; Nanda, R.; Nangia, J. R.; Storniolo, A. M.; Traina, T. A.; Vaklavas, C.; Poznak, C. H. V.; Wolff, A. C.; Forero-Torres, A.; Hayes; D. F. Significance of Circulating Tumor Cells in Metastatic Triple Negative Breast Cancer Patients within a Randomized, Phase II Trial: TBCRC 019. Clin. Cancer Res. 2015, 21, 2771-2779, DOI: 10.1158/1078-0432.CCR-14-2781. (6)

Alix-Panabieres, C; Pantel, K. Circulating Tumor Cells: Liquid Biopsy of Cancer. Clin. Chem. 2013, 59, 110-118, DOI: 10.1373/clinchem.2012.194258.

(7)

Steeg, P. S. Tumor Metastasis: Mechanistic Insights and Clinical Challenges. Nature Med. 2006, 12, 895-904, DOI: 10.1038/nm1469. ACS Paragon Plus Environment

44

Page 45 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8)


Parkinson, D. R.; Dracopoli, N.; Petty, B. G.; Compton, C.; Cristofanilli, M.; Deisserogh, A.; Hayes, D. F.; Kapke, G.; Kumar, P.; Lee, J. S. H.; McCormack, R.; Mikulski, S.; Nagahara, L.; Pantel, K.; Pearson-White, S.; Punnoose, E. A.; Roadcap, L. T.; Schade, A. E.; Scher, H. I.; Sigman, C. C.; Kelloff, G. J. Considerations in the Development of Circulating Tumor Cell Technology for Clinical Use. J. Translat. Med. 2012, 10, 138, DOI: 10.1186/1479-5876-10-138.

(9)

Ghossein, R. A.; Bhattacharya, S.; Rosai, J. Molecular Detection of micro metastases and Circulating Tumor Cells in Solid Tumors. Clin. Cancer Res. 1999, 5, 1950-1960.

(10) King, M. R. Rolling in the Deep: Therapeutic targeting of circulating tumor cells. Frontiers in Oncol. 2012, 2, 184, DOI: 10.3389/fonc.2012.00184. (11) Yu, M.; Stott, S.; Toner, M.; Maheswaran, S.; Haber, D. A. Circulating Tumor Cells: Approaches to Isolation and Characterization. J. Cell. Biol. 2011, 192, 373-382, DOI: 10.1083/jcb.201010021. (12) Lustberg, M.; Jatana, K. R.; Zborowski, M.; Chalmers, J. Emerging Technologies for CTC Detection Based on Depletion of Normal Cells. Recent Results Cancer Res. 2012, 195, 97-110, DOI: 10.1007/978-3-642-28160-0_9. (13) Hou, J. M.; Greystoke, A.; Lancashire, L.; Cummings, J.; Ward, T.; Board, R.; Amir. E.; Hughes, S.; Krebs, M.; Hughes, A.; Ranson, M.; Lorigan, P.; Dive, C.; Blackhall, F. H. Evaluation of Circulating Tumor Cells and Serological Cell Death Biomarkers in Small Cell Lung Cancer Patients

Undergoing

Chemotherapy.

Am.

J.

Pathol.

2009,

175,

808-816,

DOI:

10.2353/ajpath.2009.090078. (14) Miller, M. C; Doyle G. V; Terstappen, L. W. Significance of Circulating Tumor Cells Detected by the Cell Search System in Patients with Metastatic Breast Colorectal and Prostate Cancer. J. Oncol. 2010, 2010, 617421, DOI: 10.1155/2010/617421. (15) Beije, N; Jager, A; Sleijfer, S. Circulating Tumor Cell Enumeration by the Cell Search System: the Clinician’s Guide to Breast Cancer Treatment? Cancer Treat. Rev. 2015, 41, 144-150, DOI: 10.1016/j.ctrv.2014.12.008.


45


Page 46 of 54

(16) Yoon, H. J.; Kim, T. H.; Zhang, Z.; Azizi, E.; Pham, T. M.; Paoletti, C.; Lin, J.; Ramnath, N.; Wicha, M. S.; Hayes, D. F.; Simeone, D. M.; Nagrath, S. Sensitive Capture of Circulating Tumour Cells by Functionalized Graphene Oxide Nanosheets. Nature Nanotech. 2013, 8, 735-741, DOI: 10.1038/nnano.2013.194. (17) Ariyasu, S.; Hanaya, K.; Watanabe, E.; Suzuki, T.; Horie, K.; Hayase, M.; Abe, R.; Aoki, S. Selective Capture and Collection of Live Target Cells Using a Photoreactive Silicon Wafer Device Modified with Antibodies via a Photocleavable Linker. Langmuir 2012, 28, 13118-13126, DOI: 10.1021/la302393p. (18) Lv, S.; Wang, J.; Xie, M.; Lu, N.; Li, Z.; Cai, X.; Cai, S.; Zhang, P.; Dong, P.; Huang, W.; Photoresponsive Immunomagnetic Nanocarrier for Capture and Release of Rare Circulating Tumor Cells. Chem. Sci. 2015, 6, 6432-6438, DOI: 10.1039/C5SC01380A. (19) Myung, J. H.; Launiere, C. A.; Eddington, D. T.; Hong, S. Enhanced Tumor Cell Isolation by a Biomimetic Combination of E-selectin and Anti-EpCAM: Implications for the Effective Separation

of

Circulating

Tumor

Cells

(CTCs).

Langmuir

2010,

26,

8589-8596,

DOI: 10.1021/la904678p. (20) Lou, H. Y.; Zhao, W.; Hanson, L.; Zeng, C.; Ciu, Y.; Cui, B. Dual-Functional Lipid Coating for the Nanopillar-Based Capture of Circulating Tumor Cells with High Purity and Efficiency. Langmiur 2017, 33, 1097-1104, DOI: 10.1021/acs.langmuir.6b03903. (21) Lustberg, M. B; Balasubramanian, P; Miller, B; Garcia-Villa, A; Deighan, C; Wu, Y.; Wu, Y.; Carothers, S.; Berger, M.; Ramaswamy, R.;, Macrae, E. R.; Wesolowski, R.; Layman, R. M.; Mrozek, M.; Pan, X.; Summers T. A.; Shapiro, C. L.; Chalmers, J. J. Heterogeneous Atypical Cell Populations are Present in Blood of Metastatic Breast Cancer Patients. Breast Cancer Res. 2014, 16, R23, DOI: 10.1186/bcr3622. (22) Yu, M.; Bardia, A.; Wittner, B. S.; Stott, S. L.; Smas, M. E.; Ting, D. T.; Isakoff, S. J.; Ciciliano, J. C.; Wells, M. N.; Shah, A. M.; Concannon, K. F.; Donaldson, M. C.; Sequist, L. V.; Brachtel, E.; Sgroi, D.; Baselga, J.; Ramaswamy, S.; Toner, M.; Haber, D. A.; Maheswaran, S. Circulating ACS Paragon Plus Environment

46

Page 47 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition. Science 2013, 339, 580-584, DOI: 0.1126/science.1228522. (23) Soysal, S. D; Muenst, S; Barbie, T; Fleming, T; Gao, F; Spizzo, G.; Oertli, D.; Viehl, C. T.; Obermann, E. C.; Gillanders, W. E. EpCAM Expression Varies Significantly and is Differentially Associated with Prognosis in the Luminal B HER2(+), Basal-like, and HER2 Intrinsic Subtypes of Breast Cancer. Br. J. Cancer. 2013, 108, 1480-1487, DOI: 10.1038/bjc.2013.80. (24) Gorges, T.; Tinhofer, I.; Drosch, M.; Röse, L.; Zollner, T.; Krahn, T.; Ahsen, O. V. Circulating Tumour Cells Escape from EpCAM-based Detection Due to Epithelial-to-mesenchymal Transition. BMC Cancer 2012, 12, 178, DOI: 10.1186/1471-2407-12-178. (25) Hong, B; Zu, Y. Detecting Circulating Tumor Cells: Current Challenges and New Trends. Theranostics 2013, 3, 377-394, DOI: 10.7150/thno.5195. (26) Azarin, S. M.; Yi, J.; Gower, R. M.; Aguado, B. A.; Sulivan, M. E.; Goodman, A. G.; Jiang, E. J.; rao, S. S.; Ren, Y.; Tucker, S. L.; Backman, V.; Jeruss, J. S.; Shea, L. D. Invivo Capture and Label-free Detection of Early Metastatic Cells. Nature Commun. 2014, 6, 1-9, DOI: 10.1038/ncomms9094. (27) Sun, W.; Jia, C; Huang, T.; Li, G.; Zhang, H.; Jing, F.; Jin, Q.; Zhao, J.; Li, G.; Zhang, Z.; High-performance Size-based Microdevice for the Detection of Circulating Tumor Cells from Peripheral

Blood

in

Rectal

Cancer

Patients.

PLoS

One.

2013,

8,

e75865,

DOI:

10.1371/journal.pone.0075865. (28) Jin, C.; McFaul, S. M.; Duffy, S. P.; Deng, X.; Tavassoli, P.; Black, P. C.; Ma, H. Technologies for Label-free Separation of Circulating Tumor Cells: from Historical Foundations to Recent Developments. Lab Chip 2014, 14, 32-44, DOI: 10.1039/c3lc50625h. (29) Farace, F.; Massard, C.; Vimond, N.; Drusch, F.; Jacques, N.; Billiot, F.; Laplanche, A.; Chauchereau, A.; Lacroix, L.; Planchard, D.; Le Moulec, S.; André, F.; Fizazi, K.; Soria, J. C.; Vielh, P. A Direct Comparison of CellSearch and ISET for Circulating Tumour-cell Detection in ACS Paragon Plus Environment

47


Patients

with

Metastatic

Carcinomas.

Br.

J.

Cancer

Page 48 of 54

2011,

105,

847-853,

DOI:

10.1038/bjc.2011.294. (30) Nagrath, S.; Sequiwst, L. V.; Maheswaran, S.; Bell, D. W.; Irimia, D.; Ulkus, L.; Smith, M. R.; Kwak, E. L.; Digumarthy, S.; Muzikansky, A.; Ryan, P.; Balis, U. J.; Tompkins, R. G.; Haber, D. A.; Toner, M. Isolation of Rare Circulating Tumour Cells in Cancer Patients by Microchip Technology. Nature 2007, 450, 1235-1239, DOI:10.1038/nature06385. (31) Stott, S. L.; Hsu, C. H.; Tsukrov, D. I.; Yu, M.; Miyamoto, D. T.; Waltman, B. A.; Rothenberg, S. M.; Shah, A. M.; Smas, M. E.; Korir, G. K.; Floyd, F. P Jr.; Gilman, A. J.; Lord, J. B.; Winokur, D.; Springer, S.; Irimia, D.; Nagrath, S.; Sequist. L. V.; Lee, R. J.; Isselbacher, K. J.; Maheswaran, S.; Haber, D. A.; Toner, M. Isolation of Circulating Tumor Cells Using a Microvortex-generating Herringbone-chip.

Proc.

Natl.

Acad.

Sci.

2010,

107,

18392-18397,

DOI:

10.1073/pnas.1012539107. (32) Vona, G.; Sabile, A.; Louha, M.; Sitruk, V.; Romana, S.; Schutze, K.; Capron, F.; Franco, D.; Pazzagli, M.; Vekemans, M.; Lacour, B.; Brechot, C.; Paterlini-Brechot, P. Isolation by Size of Epithelial Tumor Cells. Am. J. Pathol. 2000, 156, 57-63, DOI: 10.1016/S0002-9440(10)64706-2. (33) Park, S.; Ang, R. R.; Duffy, S. P.; Bazov, J.; Chi, K. N.; Black, P. C.; Ma, H. Morphological Differences Between Circulating Tumor Cells from Prostate Cancer Patients and Cultured Prostate Cancer Cells. PLoS One 2014, 9, e85264, DOI: 10.1371/journal.pone.0085264. (34) Mohamed, H.; Murray, M.; Turner, J. N.; Caggana, M. Isolation of Tumor Cells Using Size and Deformation. J. Chromatogr. A. 2009, 1216, 8289-8295, DOI: 10.1016/j.chroma.2009.05.036. (35) Yusa, A.; Toneri, M.; Masuda, T.; Ito, S.; Yamamoto, S.; Okochi, M.; Kondo, N.; Iwata, H.; Yatabe, Y.; Ichinosawa, Y.; Kinuta, S.; Kondo, E.; Honda, H.; Arai, F.; Nakanishi, H. Development of a New Rapid Isolation Device for Circulating Tumor Cells (CTCs) using 3D Palladium Filter and its Application for Genetic Analysis. PLoS One 2014, 9, e88821, DOI: 10.1371/journal.pone.0088821. (36) Lin, H. K.; Zheng, S.; Williams, A. J.; Balic, M.; Groshen, S.; Scher, H. I., Fleisher, M.; Stadler, ACS Paragon Plus Environment

48

Page 49 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


W.; Datar, R. H.; Tai, Y. C.; Cote, R. J. Portable Filter-based Microdevice for Detection and Characterization of Circulating Tumor Cells. Clin. Cancer Res. 2010, 16, 5011-5018, DOI: 10.1158/1078-0432.CCR-10-1105. (37) Sarioglu, A. F.; Aceto, N.; Kojic, N.; Donaldson, M. C.; Zeinali, M.; Hamza, B.; Engstron, A.; Zhu, H.; Sundaresan, T. K.; Miyamoto, D. T.; Luo, X.; Bardia, A.; Wittner, B. S.; Ramaswamy, S.; Shioda, T.; Ting, D. T.; Stott, S. L.; Kapur, R.; Maheswaran, S.; Haber, D. A.; Toner, M. A Microfluidic Device for Label-free Physical Capture of Circulating Tumor Cell Cluster. Nature Meth. 2015, 12, 685-691, DOI: 10.1038/nmeth.3404. (38) Zheng, S.; Lin, H.; Liu, J. Q.; Balic, M.; Datar, R.; Cote, R. J.; Tai, Y. C. Membrane Microfilter Device for Selective Capture, Electrolysis and Genomic Analysis of Human Circulating Tumor Cells. J. Chromatogr. A. 2007, 1162, 154-161, DOI: 10.1016/j.chroma.2007.05.064. (39) Zheng, S.; Lin, H. K.; Lu, B.; Williams, A.; Datar, R.; Cote, R. J.; Tai, Y. C. 3D micro-filter Device for Viable Circulating Tumor Cell (CTC) Enrichment from Blood. Biomed. Microdevices 2011, 13, 203-213, DOI: 10.1007/s10544-010-9485-3. (40) Rostango, P.; Moll, J. L.; Bisconte, J. C.; Caldani, C. Detection of Rare Circulating Breast Cancer Cells by Filtration Cytometry and Identification by DNA content: Sensitivity in an Experimental Model. Anticancer Research 1997, 17, 2481-2485. (41) Hosokawa, M.; Kenmotsu, H.; Koh, Y.; Yoshino, T.; Yoshikawa, T.; Naito, T.; Takahashi, T.; Murakami, H.; Nakamura, Y.; Tsuya, A.; Shukuya, T.; Ono, A.; Akamatsu, H.; Watanabe, R.; Ono, S.; Mori, K.; Kanbara, H.; Yamaguchi, K.; Tanaka, T.; Matsunaga, T.; Yamamoto, N. Size-based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System. PLoS One 2013, 8, e67466, DOI: 10.1371/journal.pone.0067466. (42) Okano, H.; Konishi, T.; Suzuki, T.; Suzuki. T.; Ariyasu, S.; Aoki, A.; Abe, R.; Hayase, M. Enrichment of Circulating Tumor Cells in Tumor-bearing Mouse Blood by a Deterministic Lateral Displacement

Microfluidic

Device.

Biomed.

Microdevices

10.1007/s10544-015-9964-7. ACS Paragon Plus Environment

49

2015,

17,

9964,

DOI:


Page 50 of 54

(43) Deng, Y.; Zhang, Y.; Sun, S.; Wang, Z.; Wang, M.; Yi, B.; Czjkowsky, D. M.; Liu, B.; Li, Y.; Wei, W.; Shi, Q. An Integrated Microfluidic Chip System for Single-Cell Secretion Profiling of Rare Circulating Tumor Cells. Sci. Reports 2014, 4, 7499, DOI: 10.1038/srep07499. (44) Tan, S. J.; Yobas, L.; Lee, G. Y.; Ong, C. N.; Lim, C. T. Microdevice for the Isolation and Enumeration of Cancer Cells from Blood. Biomed. Microdevices 2009, 11, 883-892, DOI: 10.1007/s10544-009-9305-9. (45) Tan, S. J.; Lakshmi, R. L.; Chen, P.; Lim, W. T.; Yobas, L.; Lim, C. T. Versatile Label Free Biochip for the Detection of Circulating Tumor Cells from Peripheral Blood in Cancer Patients. Biosens. Bioelectron. 2010, 26, 1701-1705, DOI: 10.1016/j.bios.2010.07.054. (46) Bhagat, A. A.; Hou, H. W.; Li, L. D.; Lim, C. T.; Han, J. Pinched Flow Coupled Shear-modulated Inertial Microfluidics for High-throughput Rare Blood Cell Separation. Lab Chip 2011, 11, 1870-1878, DOI: 10.1039/C0LC00633E. (47) Wang, S.; Wang, H.; Jiao, J.; Chen, K.-J.; Owens, G. E.; Kamei, K.; Sun, J.; Sherman, D. J.; Behrenbruch, C. P.; Wu, H.; Tseng, H. R. Three-Dimensional Nanostructured Substrates toward efficient Capture of Circulating Tumor Cells. Angew. Chem. Int. Ed. 2009, 48, 8970-8973, DOI: 10.1002/anie.200901668. (48) Warkiani, M. E.; Khoo, B. L.; Wu, L.; Tay, A. K. P.; Bhagat, A. A. S.; Han, J.; Lim, C. T. Ultra-fast, Label-free Isolation of Circulating Tumor Cells from Blood Using Spiral Microfluidics, Nature Protocols 2016, 11, 134-148, DOI: 10.1038/nprot.2016.003. (49) Shashni, B.; Ariyasu, S.; Takeda, R.; Suzuki, T.; Shiina, S.; Akimoto K.; Maeda, T.; Aikawa, N.; Abe, R.; Osaki, T.; Itoh, N.; Shin, A. Size-based Differentiation of Cancer and Normal Cells by a Particle Size Analyzer Assisted by a Cell-recognition PC Software. Biol. Pharm. Bull. 2018, 41, 487-503. (50) It should be mentioned that the “number” of the captured and recovered cancer cells is more important than the recovery percentage (%). Each GBF has its capacity for the capture of cancer cells and we assume that it is big enough to trap very small number of CTCs in the real blood ACS Paragon Plus Environment

50

Page 51 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


samples of cancer cells e.g. in single filtration and circulation filtration, we recovered a total of ca. 2.5 × 106 cells (filtration volume=5 mL) and ca. 50 × 106 cells (filtration volume=50 mL), respectively, which are greater than the reported CTCs in the human blood (ca. 10 CTC/mL; 0.05 × 106 CTCs in human body (10 CTCs × 5000 mL of blood)). (51) It is considered that there are air layers and/or bubbles between three 0.4 mm GBFs, which hamper the smooth filtration of non-diluted blood. These air layers and/or bubbles could be easily removed when diluted blood was filtered. Therefore, the use of single (one layer) GBF (thickness should be adjusted for experimental and clinical purposes) is recommended for the filtration of non-diluted blood containing CTCs. (52) In trial experiment for Figure S17 in Supporting Information, LLC-EGFP (1 × 106) cells were injected to C57 mouse and the blood was collected after circulation for 30 min and 60 min, filtered through GBF, and cultured on/in GBF for 5 days. After these procedures, 30 min and 60 min samples showed very few and negligible GFP-positive cells (data not shown), respectively (even after incubation for 5 days). Therefore, we concluded that it is unlikely that the injected LLC-EGFP cells are still circulating in the blood even 13 days after inoculation in the mice and are collected by GBF.


51


TABLE OF CONTENTS (TOC) GRAPHIC

Simple and Convenient Method for the Isolation, Culture, and Recollection of Cancer Cells from Blood by Using Glass-Beads Filters

Babita Shashni, Hidehiko Matsuura, Riku Saito, Takuma Hirata, Shinya Ariyasu, Kenta Nomura, Hiroshi Takemura, Kazunori Akimoto, Naoyuki Aikawa, Atsuo Yasumori, and Shin Aoki


52

Page 52 of 54

Page 53 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



53


Capture, Growth and Recollection of Cancer Cells using Glass-beads Filter 207x119mm (300 x 300 DPI)


Page 54 of 54

Simple and Convenient Method for the Isolation, Culture, and

Recommend Documents