AU2021101054A4 - Method for Efficiently Separating Neutrophils from Human Solid Tumors - Google Patents

Abstract

The present invention discloses a method for efficiently separating neutrophils from human solid tumors, comprising the step of obtaining neutrophils by preparing a single cell suspension through papain-mediated separation of tumor tissues, and analyzing the single cell suspension based on the CD66B-mediated flow cytometry. According to the present invention, neutrophils are separated from glioma tissues by combining the step of papain-mediated tissue dissociation and CD66B-mediated FACS in the separation process of GANs. Test proves that the average number of living cells separated from glioma tissues under the mediation of papain reaches 6.3x106 cells/g, and 97% of CD66B is retained after glioma tissues are digested with papain for 15 min. Thus, the separation method of the present invention enables efficient separation of neutrophils in solid tumors and provides technical support for studying patient-derived tumor-associated immune cells and their biological functions in the progression and relapse of solid tumors. 2/5 A B Ceramic Beads Enzymic Digest Cut Triturt Cut Filtar Count Erythrocyte Dead Cet Filter Count Erythrocyte Dead Cells lnacuvate Lysis Remove Lysis Remove isGliuma Tissue 0 Leukocyte S Erythrocyte S Gl1iomarell Deoxyribonuclease Trypsirior Papain i Qvomucoid Inhibitor-Abtjrnin C * D PeripheralBlood 15Cmri~ n15min 30mri 45Smin 8 0.7 7837(. T 75.5 0* 4 IN IN E Op A fl a S -~L 66s4. CD66 S * IChi-Sqarel1.767, I-8 Chi-Squara=4.722. p-0,029 Chi-Square=5.OO, '' p-O.O2S Fig. 2

Description

2/5

A B Ceramic Beads Enzymic Digest

Cut Triturt Cut Filtar

Count Erythrocyte Dead Cet Filter Count Erythrocyte Dead Cells lnacuvate Lysis Remove Lysis Remove

isGliuma Tissue 0 Leukocyte S Erythrocyte S Gl1iomarell

Deoxyribonuclease Trypsirior Papain i Qvomucoid Inhibitor-Abtjrnin

C * D PeripheralBlood

15Cmri~n15min 30mri 45Smin 80.7 7837(. T 75.5 0* 4 IN IN

E Op A

fl a

S -~L 66s4. CD66 S* IChi-Sqarel1.767, I-8 Chi-Squara=4.722. p-0,029

Chi-Square=5.OO, '' p-O.O2S

Fig. 2

Method for Efficiently Separating Neutrophils from Human Solid Tumors

TECHNICAL FIELD

The present invention relates to the technical field of biomedicines, in

particular to a method for efficiently separating neutrophils from human solid

tumors.

BACKGROUND

Immunocytes play a dual role in tumor progression. In the spontaneous

immune system, tumor-associated gene mutations produce multiple

neoantigens such as EGFRvIII, bcr-abl, ERG and LMP2, which are absorbed

by antigen-presenting cells (e.g. dendritic cells, monocytes and B

lymphocytes), presented by MHC-II complex, and then recognized by T cell

antigen receptor (TCR) to activate T or B lymphocytes. Perforin, serine ester

and lymphotoxin produced by the activated CD8+T cells can kill tumor cells;

the antigen-specific antibodies, and IL-2 and other interleukins produced by

the activated B cells can regulate the immune surveillance of tumors; IFN and

IL-2 secreted by T and B lymphocytes can recruit and activate natural immune

cells such as NK cells, which produce TNF and granzyme B to directly kill

tumor cells. These spontaneous immune processes can inhibit the growth and

progression of tumors. In the tumor-adaptive immune system, the highly

evolved cancer cells such as tumor stem cells can secrete CCL2, IL-6 and IL

, and recruit a variety of immunosuppressive regulatory cells such as regulatory T cells and myeloid-derived suppressor cells to suppress the immune response induced by cytotoxic CD8+T cells, B cells and NK cells. In addition, the evolved cancer cells directly change the fate of several innate immune cells such as malignant tumor cells secreting IL-4, IL-13, IL-17 and

TGF, which polarize the tumor-associated macrophages into tumor-promoting

phenotype. These tumor-associated immune cells induce tumor immune

escape, and promote tumor growth, angiogenesis, invasion and metastasis,

resulting in a malignant phenotype. These findings expand our understanding

of tumor biology, and provide prospects for the development of tumor

immunotherapy. However, our current understanding of immune cell

differentiation is based on the mouse models, and the functional role of

immune cells in human-derived tumor tissues remains to be investigated.

As the most abundant leukocytes in the circulation, neutrophils are

traditionally considered to defend the host against microbial invasion and aid

in wound healing. However, a wealth of data suggests that neutrophils play a

key role in tumor progression. For example, neutrophil extracellular trap

(NET)-associated protease can cleave laminin, and the proteolytically

remodeled laminin activates the integrin signaling pathway, thereby inducing

proliferation of dormant cancer cells. In addition, NET-DNA can be sensed by

transmembrane receptor CCDC25, which activates ILK-parvin pathway and

promotes tumor cell metastasis. Furthermore, both surgery and radiotherapy

can induce the infiltration of Ly6G+ neutrophils into the tumor area. The

surgically recruited neutrophils are able to cause glioma cell death, which

leads to local necrosis and consequently tumor progression. The

radiotherapy-recruited neutrophils are capable of regulating the inverse differentiation of glioma cells into glioma stem cells via NOS2-NO-ID4.

However, these findings on neutrophil function are mainly from mice. Due to

the short life span of neutrophils in vitro, the physiological function of glioma

associated neutrophils (GANs) from patients is unclear. It is still a difficult

problem to separate neutrophils from solid tumors.

Neutrophils in peripheral blood are usually separated by Ficoll, a method

based on the density gradient centrifugation. However, this method may not

be suitable for separating neutrophils from solid tumors because the cellular

composition in tumor tissues is more complex than that in blood. Flow

cytometer can separate or monitor specific lineage cells according to multiple

fluorescence signals generated by individual fluorescence-binding antigen

antibody reactions. Therefore, fluorescence-activated cell sorting (FACS) is

commonly applied to separate immune cells from whole blood by multiple

combinations of surface markers. In solid tumors, the first step in separating

GANs is tissue separation, a process that leads to physical or chemical

disruption of cell surface proteins, including cell-lineage-specific markers, to

the detriment of surface antigen-mediated isolation. Therefore, a suitable

method for separating solid tumors needs to be explored.

SUMMARY

Aiming at solving the problems in the prior art, the purpose of the present

invention is to provide a method for efficiently separating glioma-associated

neutrophils, which achieves the efficient separation of patient-derived tumor

neutrophils by combining papain-mediated separation of tissues and CD66B- mediated flow cytometry.

In order to achieve the above purpose, the technical solutions used in the

present invention are as follows:

The present invention provides a method for efficiently separating

neutrophils from human solid tumors, comprising the step of obtaining

neutrophils by preparing a single cell suspension through papain-mediated

separation of tumor tissues, and analyzing the single cell suspension based

on the CD66B-mediated flow cytometry.

Preferably, the papain-mediated separation of tumor tissues for the

preparation of a single cell suspension specifically comprises the following

methods and steps:

adding a mixing solution of deoxyribonuclease and papain to a tumor,

mixing, incubating at 37 °C for 1-45 min, centrifuging and collecting cell

granules; and

resuspending the cell granules in ovomucin inhibitor-albumin, treating

with a debris removal solution, centrifuging, collecting the precipitate and

lysing with a mixing solution of PBS and sterile water to obtain a single cell

suspension.

Preferably, the mixing solution of deoxyribonuclease and papain is

obtained by mixing deoxyribonuclease and papain according to the volume

ratio of 1:1-1:20 (preferably 1:10);

the mixing solution of deoxyribonuclease and papain is added to the

tumor at a concentration of 100-500 pL/g (preferably 300pL/g) according to the tumor mass.

Preferably, the centrifugation condition is as follows: centrifuging at 1000

rpm for 5 min.

Preferably, the mixing solution of PBS and sterile water is obtained by

mixing PBS and sterile water according to the volume ratio of 1:1-1:5.

Preferably, the CD66B-mediated flow cytometry analysis of single cell

suspension comprises the following steps: centrifuging the prepared single

cell suspension, collecting cell granules, resuspending the cell granules in

FBS-PBS solution, blocking with human BD Fc blocker for 10 min at room

temperature, sequentially washing, staining and re-washing the cells,

detecting the CD66B*/CD15+ cell proportion by flow cytometer, and sorting

CD66B+ neutrophils.

Preferably, the centrifugation condition is as follows: centrifuging at 1500

rpm for 5 min.

Preferably, the solid tumor comprises a glioma.

The prevent invention discloses the following technical effects:

According to the method for efficiently separating neutrophils from human

solid tumors disclosed herein, the conventional separation of neutrophils is

mostly implemented in mouse models or in HL-60 (human acute

promyelocytic leukemia cell line) differentiated neutrophil-like cells. The

present invention combines a step of papain-mediated tissue dissociation and

CD66B-mediated FACS in the separation process of GANs. Test proves that

the average number of living cells separated from glioma tissues under the mediation of papain reaches 6.3x106 cells/g, and 97% of CD66B is retained after glioma tissues are digested with papain for 15 min. It is thus clear that this method can successfully separate glioma-associated neutrophils, improve cell vitality, maintain neutrophil surface antigen, and enhance the interaction between antigen and antibody, which provide technical support for further understanding the biological function of human-derived tumor infiltrating neutrophils.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain more clearly the embodiments in the present invention

or the technical solutions in the prior art, the following will briefly introduce the

drawings needed in the description of the embodiments. Obviously, drawings

in the following description are only some embodiments of the present

invention, and for a person skilled in the art, other drawings may also be

obtained based on these figures without paying any creative effort.

Fig. 1 shows the distribution of neutrophils in human glioma tissues

assessed by hematoxylin-eosin staining of different grades of glioma tissues;

Figs. A-D show the infiltration of neutrophils in different grades of glioma

tissues; and Fig. E shows the percentage of neutrophils in glioma tissues of

different grades.

Fig. 2 compares the separation effect of glioma tissues digested with

papain and by physical grinding; Fig. A is a schematic diagram of glioma

tissues treated by the physical grinding method; Fig. B is a schematic diagram of glioma tissues digested by papain; Fig. C shows the number of living cells after glioma tissues are physically ground and digested with trypsin and papain; and Fig. D shows the comparison of the percentage of CD66B+ cells after digestion with papain for different time.

Fig. 3 shows the screening of isolated surface markers suitable for GANs;

Fig. A shows the results of the flow cytometry by co-labeling neutrophils with

CD15 and CD66B; Fig. B shows the immunofluorescence assay of

neutrophils in glioma tissues assessed by combining CD44 and CD66B; Fig.

C shows the distribution percentage of CD44+ cells; Fig. D shows the

distribution of neutrophils, eosinophils and basophils; and Fig. E shows the

distribution percentage of neutrophils.

Fig. 4 shows the purification analysis of glioma-associated neutrophils

(GANs) by different isolation methods; Fig. A is a schematic diagram of two

purification methods, i.e. Ficoll-mediated density gradient centrifugation and

FACS; Fig. B shows the purification result of blood-derived neutrophils by

Ficoll; Fig. C shows the purification result of glioma tissue-derived

neutrophils by Ficoll; Fig. D shows the analysis of purification results of

glioma tissue-derived neutrophils after CD66B-mediated FACS separation;

Fig. E shows the quantitative real-time PCR identification results of FACS

sorted glioma tissue-derived CD66B+ cells (TP); and Fig. F is the expression

of neutrophil marker genes CEACAM8, ITGAM, FUT4, PTPRC and CD44 in

different TP, TN and BP populations.

Fig. 5 shows the distribution of basophils in glioma tissues.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be

described in detail, which should not be construed as being limited thereto,

but should be understood as a more detailed description of certain aspects,

features and embodiments thereof.

It should be understood that the terms described herein are only intended

to describe specific embodiments, and are not intended to limit the present

invention. Furthermore, the range of values in the present invention should be

such understood that each intermediate value between the upper and lower

limits of the range is also specifically disclosed. Each smaller range between

any stated value or intermediate value within a stated range and any other

stated value or intermediate value within a stated range is also included in the

present invention. The upper and lower limits of these smaller ranges can be

independently included in or excluded from the scope.

Unless otherwise indicated, all technical and scientific terms used herein

have the same meaning as commonly understood to one of ordinary skill in

the art to which this prevent invention belongs. Although the present invention

describes only preferred methods and materials, any methods and materials

similar or equivalent to those described herein can be used in the practice or

testing of the prevention invention.

It will be readily apparent to those skilled in the art that various

modifications and changes can be made to the specific embodiments of the

specification of the prevent invention without departing from the scope or spirit

of the prevent invention. Upon reading this disclosure, many alternative embodiments of the present invention will be apparent to persons of ordinary skill in the art. The specification and examples of this application are only exemplary.

As used herein, the terms "including", "comprising", "having" and

"containing" are all open terms, which means including but not limited to.

Unless otherwise specified, the reagents or materials used in the

following examples can be obtained through commercial channels.

Example 1 Method for efficiently separating neutrophils from glioma

1. Test materials

1.1. Human tissue specimens and peripheral blood

Tissue specimens were obtained from patients who had underwent

surgical resection after pathological diagnosis, including 2 grade II glioma

specimens, 3 grade III glioma specimens, 8 grade IV glioma specimens and 1

leptomeningioma specimen (many granulocytes infiltrated). Of them, 8 normal

brain specimens were obtained from donors with craniocerebral trauma. The

matched peripheral blood was venous blood of glioma patients. The study

was approved by Tianjin Cancer Research Institute and Ethics Committee of

Tianjin Cancer Hospital, and the written informed consent of each patient was

provided.

1.2 Hematoxylin-eosin staining

Glioma specimens were fixed with formalin and embedded in paraffin.

The paraffin blocks were cut into paraffin sections, which were dewaxed,

hydrated, stained with hematoxylin for 5 min and eosin for 3 min, and then soaked in ethanol and xylene sequentially. Finally, the tissue sections were covered with synthetic resin and dried in a fume hood.

2. Test method

2.1 Preparation of single cell suspension from glioma tissues

Glioma tissues were weighed and randomly divided into two groups.

Glioma specimens were cut into 0.5-1 mm 3 pieces with a scalpel. The two

groups of separated tumor pieces were made into a single cell suspension

according to the following different methods: papain was preheated at 37 °C

for 10 min, and then deoxyribonuclease (Worthington, #LK003170) and

papain (Worthington, #LK003176) were mixed into glioma tissues according

to the volume ratio of 1:10. The tissues were gently mixed with digestive juice,

and incubated at 37 °C for 15-45 min. Then, the cell suspension was filtered

by a cell sieve and centrifuged at 1000 rpm at room temperature for 5 min to

obtain the cell granules, which were resuspended in 1 mL of ovomucin

inhibitor-albumin (Worthington, #LK003182) to inactivate papain (see Fig. 2B),

and treated with the debris removal solution (Miltenyi, #130-109-398). The

obtained cell suspension was digested by papain to remove dead cells and

centrifuged at 1000 rpm for 5 min, and the erythrocytes were lysed with a

mixing solution of PBS and sterile distilled water (VV=1:3).

Meanwhile, the physical grinding-based tissue separation method was

taken as a comparative example as follows: transferring the glioma specimen

sections into a 1.5 mL centrifuge tube, soaking in 1 mL of PBS, then

homogenizing with sterilized ceramic balls for 10 s, and filtering the cell

suspension to remove the tissue mass (see Fig. 2A); removing dead cells by physically grinding the obtained cell suspension, centrifuging at 1000 rpm for min, and then lysing erythrocytes with a mixing solution of PBS and sterile distilled water (VN=1:3).

2.2 Preparation of peripheral blood leukocyte suspension

Peripheral blood was mixed with erythrocyte sedimentation (TBD, #HES

TBD-550) and allowed to stand at room temperature for 30 min. The

supernatant was then transferred to a clean 15 mL centrifuge tube and the

cells were centrifuged and pelleted at 1000 rpm for 5 min, and the

erythrocytes were lysed with a mixing solution of PBS and sterile distilled

water (VN=1:3).

2.3 Fluorescence-activated cell sorting (FACS)

The single cell suspension was centrifuged at 1500 rpm for 5 min, and

then the cell granules were resuspended in 2% FBS-PBS solution (2% FBS

was added to PBS). The samples were blocked with 5 pL of human BD Fc

blocker (BD, #564219) at room temperature for 10 min and washed with 1 mL

of 2% FBS-PBS solution. The samples were stained with anti-human CD66B

FITC (Biolegend, #305104) or anti-human CD15-APC (Biolegend, #301908)

respectively according to the test, and stored on ice for 20 min. The stained

cells were washed again, and the cell proportion of CD66B+/CD15+ was

detected by flow cytometry or CD66B+ cells were sorted. The data were

analyzed by FlowJO7.6.

2.4 Immunofluorescence assay

Paraffin-embedded glioma sections were dewaxed and hydrated, and then sealed with a blocking buffer containing 5% BSA at room temperature for 30 min. After being immunolabeled with the primary antibodies of anti

CD66B (1:100, Novus Biologicals, #NB100-64916), CD44 (1:100, BD,

#550392), FceR1 (1:100, Invitrogen, #12-5899) and SIGLEC8 (1:100,

Invitrogen, #PA5-11681) separately, the sections were washed three times

with PBST and then incubated with fluorescent secondary antibody for 2 h at

room temperature and protected from light. Nucleuses were re-stained with

DAPI. Immunofluorescence images were collected by a Zeiss fluorescence

microscope.

2.5 Purification of granulocytes by Ficoll

Human neutrophil separation solution (Solarbio, #P2410/P9040) was

added to a clean 15 mL centrifuge tube. Then the single cell suspension was

added dropwise above the separation solution and centrifuged at 800 g for 20

min at room temperature, which rendered three distinct liquid phases:

lymphocytes in the top layer, granulocytes in the middle layer and

erythrocytes in the bottom layer. The granulocytes were carefully transferred

into a clean centrifuge tube and washed with PBS.

2.6 Quantitative RT-PCR

Total RNA of isolated cells was extracted with TRIzol reagent (Invitrogen,

#15596018), and reversely transcribed into cDNA by using UElris RT-PCR

system (US. Everbright, #R2020-100T) for first strand cDNA synthesis.

Quantitative RT-PCR was performed on an Applied Biosystems 7500 real

time PCR system by using the Fast Super EvaGreen Qpcr master mix (U.S.

Everbright, #S2008-500T). Each quantitative RT-PCR was separately performed at least three times independently. The expression levels of associated genes were corrected by GAPDH [Chromosome 12,

NC_000012.12(6534517..6538371)]expression.

The gene-specific primers for quantitative RT-PCR are as follows:

GAPDH forward 5'-GGAGCGAGATCCCTCCAAAT-3' and reverse 5'

GGCTGTTGTCATACTTCTCATGG-3';

CEACAM8 (nucleotide sequence as shown in SEQ ID NO: 1) forward 5'

CTCTAATCCACCCTCCAGTATTC-3' and reverse 5'

AGGATCCGCTGTTCTTTGGTAG-3';

ITGAM (nucleotide sequence as shown in SEQ ID NO: 2) forward 5'

ACTTGCAGTGAGAACACGTATG-3' and reverse 5'

TCATCCGCCGAAAGTCATGTG-3';

FUT4 (nucleotide sequence as shown in SEQ ID NO: 3) forward 5'

TTGGGACCTCCTAGTTCCAC-3' and reverse 5'

TGTAAGGAAGCCATTGGA-3';

CD44 (nucleotide sequence as shown in SEQ ID NO: 4) forward 5'

ACCACAAGTTTACTAACGCAAGT-3' and reverse 5'

TCACTAATAGGGCCAGCCTC-3';

PTPRC (nucleotide sequence as shown in SEQ ID NO: 5) forward 5'

TTTGAGGGGGATTCCAGGTAAT-3' and reverse 5'

GCTCCTCAGCTTGCAGAT-3'.

The specific operation is as follows:

1) Synthesis of cDNA:

Kit: UElris II RT-PCR System for First-Strand cDNA Synthesis (with

dsDNase), US. EVERBRIGHT, #R2028.

The reverse transcription system was prepared in a nuclease-free

centrifuge tube in an ice bath, as shown in Table 1, and then gently mixed and

centrifuged instantaneously.

Table 1

Component Volume UElris II RT MasterMix(5X) 4 pL Template RNA 50 ng-1 pg dsDNase 1 pL RNase-free water Add to 20 pL Reverse transcription reaction process:

(1) incubating at 37 °C for 2 min to remove genome pollution;

(2) incubating at 55 °C for 10 min; and

(3) incubating at 85 °C for 10 s.

The obtained reverse transcription reaction products were quickly placed

on ice or stored at -20 °C.

2) Real-time PCR reaction:

Kit: Fast Super EvaGreen@qPCR Master Mix, Biocell, #S2008-500T.

Specific methods are as follows:

() melting each component of the kit in an ice bath, preparing a reaction system according to the proportion shown in Table 2, mixing gently, and centrifuging instantaneously;

Table 2

20 pL Reaction Final Reaction Components System Concentration 2xFast Super EvaGreen@Master 10pL 1x Mix F, R primer (10 pM) 1pL 0.1-0.5pM each Template 1pL 50ng 1xROX 1pL H20 Add to 20 pL

@completing the reactions by ABI 7500 according to the conditions

shown in Table 3; and

Table 3

Procedure Temperature Time Cycle Enzyme activation 950 C 2min 1 Denaturation 950 C 15s 45 Annealing & extension 600 C 60s

@ obtaining Ct value and solubility curve.

2.7 Statistical analysis

All grouped data were expressed as mean standard error of mean

(SEM). T-test was performed by Prism software (v8, GraphPad) to analyze

the statistical significance among groups. Chi-square test was carried out by statistical product and service solution (SPSS software). P<0.05 was considered statistically significant (*P<0.1, **P<0.05, ***P<0.01,

****P<0.0001). The present invention complies with the principle of

randomization.

3. Results and analysis

3.1 Selective infiltration of neutrophil-like cells into high-grade diffuse

glioma

To assess the distribution of neutrophils in human glioma tissues, the

inventors observed cells containing pea-like phyllodes (indicated by arrows in

Figs. 1C and D) in hematoxylin-eosin stained WHO grade II (n=2), grade III

(n=3), grade IV (n=8) glioma tissues and normal brain tissues (n=8), and

found that neutrophil-like cells were infiltrated in WHO grade III (Fig. 1C) and

IV (Fig. 1D) glioma tissues rather than WHO grade II (Fig. 1B) glioma tissues

and normal brain tissues (Fig. 1A).

Specifically, 0.7% of cells in grade III glioma tissues showed neutrophil

like nuclei per 2.5x103 mm2 tissue area (Fig. 1E), whereas 1.9% of cells in

grade IV glioma tissues showed a neutrophil-like nuclear phenotype in the

same area (Fig. 1E). Statistically, the percentage of neutrophil-like cells was

significantly higher in WHO IV glioma tissues than in WHO III glioma tissues

(**P<0.05) (Fig. 1E).

These results indicate that the amount of neutrophil-like cell infiltration is

positively correlated with glioma grade.

3.2 Papain effectively separates active single cells from glioma tissues

The first step to obtain tumor cells or interstitial cells from solid tumors is

tissue isolation, which methodologically usually involves disrupting cell-to-cell

contact by physical or chemical separation. Grinding, as a typical method of

physical dissociation, allows rapid homogenization of tissues and maintains

the original physiological state of dissociated single cells. Trypsin is a typical

chemical tool commonly used to separate adherent cells, and papain is widely

used to separate neurocytes from gliocytes in brain tissues.

To evaluate the advantages of papain in the separation of primary glioma

tissues, high-grade glioma specimens were firstly collected and randomly

divided into three groups. One group was physically ground with ceramic

beads (Fig. 2A), and the other two groups were digested with trypsin and

papain, respectively (Fig. 2B). The number of living cells in the single cell

suspension was counted by the trypan blue staining. It was found that the

average number of living cells produced by physical grinding was about

1.8x106 cells/g (n=4), the average number of living cells produced by trypsin

digestion was 5.5x10 6 cells/g (n=4), and the average number of living cells

produced by digestion with papain was 9.2x106 cells/g (n=4). The statistical

results showed that the method of digestion with papain produced 5.1 times

more living cells than physical grinding (**P<0.05) and 1.7 times more than

trypsin digestion (*P<0.1) (Fig. 2C). These results indicate that the method of

digestion with papain maintains cell activity and is more suitable for

separating glioma tissues.

To further investigate whether papain could destroy the protein on the

surface of tumor tissue cells, the inventors obtained isolated cells by continuously treating tumor tissues with papain, and used the flow cytometer to test the antigenicity of isolated cells obtained at four different time points of papain treatment: 0 min, 15 min, 30 min, and 45 min. The inventors analyzed the isolation rate of neutrophils and positive rate of CD66B (a common surface protein of neutrophils) according to the FSC-SSC plot of leukocytes.

All papain-treated groups and untreated groups were analyzed for CD66B+

cell separation rates by taking unstained samples as negative controls. The

results revealed that the percentage of CD66B cells in the active cell

population was 80.7%, 78.3%, 76.7% and 76.6% in the 0, 15, 30 and 45 min

groups, respectively. Compared with the 0 min group, the percentage of

CD66B cells was not statistically different in the 15 min group (chi

square=1.767, p=0.18 4 ), but a slight decrease in the 30 min group (chi

square=4.722, p=0.0 2 9) and the 45 min group (chi-square=5.005, p=0.0 2 5)

(Fig. 2D ). According to these results, tissues are digested with papain for a

short time (15 min), which may result in a slight decrease in the measured

fluorescence intensity, but hardly affect the isolation rate of CD66B+ cells, and

this treatment duration is sufficient to separate glioma tissue into single cells.

In conclusion, the test demonstrates that papain-mediated separation of solid

tumor tissues can maintain cell viability and integrity of cell surface protein.

3.3 CD66B plays a unique role as a surface marker in the separation of

GANs

Different immune cells may express the same surface proteins. For

example, CD15 and CD66B are generally expressed in neutrophils and

eosinophils, while CD44 is highly expressed in T cells and neutrophils (Table

4). These surface proteins are usually bound to recognize neutrophils in

peripheral blood. In glioma tissues, the blood-brain barrier (BBB) of brain

protection system selectively restricts the entry of certain immune cells (T

cells, B cells and NK cells). To explore which combination could be used for

the separation of GANs, the inventors analyzed the blood or glioma tissue

derived single cell suspension which was co-stained with APC-conjugated

anti-CD15 antibody and FITC-conjugated anti-CD66B antibody by flow

cytometry. The results revealed that the blood-derived living cells were mainly

divided into CD15'CD66B+ cell population and CD15-CD66B- cell population,

of which CD15+CD66B+ cells (neutrophils and eosinophils) accounted for

76.5%. Neither CD15 nor CD66B could distinguish between neutrophils and

eosinophils. Unlike peripheral blood, glioma tissue-derived living cells were

mainly divided into CD15+CD66B+ cell population, CD15+CD66B- cell

population and CD15-CD66B- cell population. CD15+CD66B+ cells accounted

for 35.4% and all CD66B+ cells allowed CD15 to be highly expressed (Fig.

3A). Apparently, CD15 hardly recognized GANs as CD15 was expressed not

only in neutrophils but also in macrophages and glioma cells in glioma tissues

(Table 4). CD66B could label CD15+CD66B+ cells alone.

Table 4

T B NK Granulocytes Glioma celcell s cells EosinophilsNeutrophilsBasophils cells

CD15 - - - - + + - +

CD44 + - - - - + - +

CD66B - - - - + + -

SIGLEC8+ - - - + - - -

FceR1 - - - - - - +

To further confirm these results, the inventors combined CD44 and

CD66B to assess neutrophils in glioma tissues. Immunofluorescence assay

clearly showed that CD66B*CD44+ cells were infiltrated in high-grade glioma

(indicated by white arrows in Fig. 3B). It was also found that up to 57.9% of

the cells were CD44+ cells per 2.5x10- 3mm 2 tissue area (Fig. 3C), mainly

because CD44 was expressed in both neutrophils and glioma cells in glioma

tissues (Table 4). CD66B+ cells accounted for 3.3% of the tissue area, and all

CD66B+ cells allowed CD44 to be highly expressed. In view of the fact that

CD44 was expressed in neutrophils but not in eosinophils (Table 4), it was

expected that eosinophils were rarely infiltrated in glioma tissues, and CD66B

exhibited specific recognition to neutrophils. To confirm this, anti-CD66B

antibody was firstly co-stained with anti-SIGLEC8 (eosinophil-specific surface

marker) antibody or anti-FceRl (basophil-specific surface marker) antibody in

human leptomeningioma tissues infiltrated by neutrophil, eosinophil, and

basophil, as confirmed pathologically. According to the pathological diagnosis,

we tested neutrophils (CD66B*SIGLEC8- cells, indicated by white arrows),

eosinophils (CD66B*SIGLEC8* cells, indicated by black arrows) (Fig. 3E) and

basophils (FceRl+ cells, indicated by arrows) in the specimens (Fig. 5). The

same tests were then performed on glioma tissues with these specific

antibodies to distinguish neutrophils. Immunofluorescence assay clearly

showed that neutrophils (CD66B*SIGLEC- cells, Fig. 3D, white arrow) were

infiltrated in high-grade glioma. In glioma tissues, eosinophils (CD66B

SIGLEC8* cells, Fig. 3D) and basophils (FceR1* cells, Fig. 5) were rarely seen. The inventors found that the percentage of neutrophils (CD66B

SIGLEC8' cells) per 2.5x10-3 mm2 tissue area was 4.8%, and no eosinophils

(CD66B-SIGLEC8* cells) were detected (Fig. 3E). These results confirm that

eosinophils are rarely infiltrated into glioma, and CD66B can be used as an

independent surface marker for the separation of GANs.

3.4 CD66B-mediated flow cytometry greatly improves the purity of

isolated GANs

In terms of mechanism, the common purification methods for tissue

infiltrated immune cells include Ficoll based on density gradient centrifugation

and FACS (Fig. 4A). To investigate the effectiveness of FACS of the present

invention for neutrophil purification, the inventors performed the separation

and analysis step by Ficoll for a comparative purpose.

Neutrophils were firstly separated from human peripheral blood by Ficoll.

The collected cells were further stained with FITC-coupled anti-CD66B

antibody and analyzed by flow cytometry. The results revealed that the purity

of CD66B+ cells was only 58.4% (Fig. 4B). To further determine the

purification ability of Ficoll in separating GANs, the inventors digested glioma

tissues with papain and separated neutrophils by Ficoll. The purity of

separated CD66B+ cells was still only 45.2% (Fig. 4C). These results confirm

the low purity of neutrophils separated by Ficoll, because many cells

(especially heterogeneous tumor cells) have a wide range of densities. The

inventors performed FACS on GANs separation, and found that the purity of

CD66B+ cells in the living cell population was only 10.4% before sorting. The

purity of CD66B+ cells was greatly increased to 100% after separated by

FACS under the mediation of CD66B (Fig. 4D), indicating that the purity of

neutrophils separated by FACS under the mediation of CD66B was higher

than that of neutrophils separated by Ficoll.

To further confirm that the separated CD66B+ cells were indeed

neutrophils, the inventors performed quantitative real-time PCR on FACS

sorted glioma tissue-derived CD66B+ cells (TP), taking glioma tissue-derived

CD66B- cells (TN) as negative control and matched peripheral blood-derived

CD66B+ cells (BP) as positive control (Fig. 4E). The results showed that the

neutrophil marker genes CEACAM8, ITGAM, FUT4, PTPRC and CD44 were

highly expressed in TP and BP populations than in TN population (Fig. 4F).

Therefore, these data indicate that the FACS-sorted CD66B+ populations are

neutrophils.

These results indicate that CD66B-based flow cytometry can more

accurately label neutrophils and improve the purity of GANs separation for

glioma tissues containing a large number of heterogeneous cells.

The preferred embodiments described herein are only for illustration

purpose, and are not intended to limit the present invention. Various

modifications and improvements on the technical solution of the present

invention made by those of ordinary skill in the art without departing from the

design spirit of the present invention shall fall within the protection scope as

claimed in claims of the present invention.

Claims (8)

1. A method for efficiently separating neutrophils from human solid

tumors, characterized by comprising the step of obtaining neutrophils by

preparing a single cell suspension through papain-mediated separation of

tumor tissues, and analyzing the single cell suspension based on the CD66B

mediated flow cytometry.

2. The method for efficiently separating neutrophils from human solid

tumors according to Claim 1, characterized in that the papain-mediated

separation of tumor tissues for the preparation of a single cell suspension

specifically comprises the following methods and steps:

adding a mixing solution of deoxyribonuclease and papain to a tumor,

mixing, incubating at 37 °C for 1-45 min, centrifuging and collecting cell

granules; and

resuspending the cell granules in ovomucin inhibitor-albumin, treating

with a debris removal solution, centrifuging, collecting the precipitate and

lysing with a mixing solution of PBS and sterile water to obtain a single cell

suspension.

3. The method for efficiently separating neutrophils from human solid

tumors according to Claim 2, characterized in that the mixing solution of

deoxyribonuclease and papain is obtained by mixing deoxyribonuclease and

papain according to the volume ratio of 1:1-1:20;

the mixing solution of deoxyribonuclease and papain is added to the

tumor at a concentration of 100-500 pL/g according to the tumor mass.

4. The method for efficiently separating neutrophils from human solid

tumors according to Claim 2, characterized in that the centrifugation condition

is as follows: centrifuging at 1000 rpm for 5 min.

5. The method for efficiently separating neutrophils from human solid

tumors according to Claim 2, characterized in that the mixing solution of PBS

and sterile water is obtained by mixing PBS and sterile water according to the

volume ratio of 1:1-1:5.

6. The method for efficiently separating neutrophils from human solid

tumors according to Claim 1, characterized in that the CD66B-mediated flow

cytometry analysis of single cell suspension comprises the following steps:

centrifuging the prepared single cell suspension, collecting cell granules,

resuspendig the cell granules in FBS-PBS solution, blocking with human

BDFc blocker for 10 min at room temperature, sequentially washing, staining

and re-washing the cells, detecting the CD66B*/CD15+ cell proportion by flow

cytometer, and sorting CD66B+ neutrophils.

7. The method for efficiently separating neutrophils from human solid

tumors according to Claim 6, characterized in that the centrifugation condition

is as follows: centrifuging at 1500 rpm for 5 min.

8. The method for efficiently separating neutrophils from human solid

tumors according to Claim 1, characterized in that the solid tumor comprises a

glioma.

FIGURES OF THE SPECIFICATION

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Fig. 1