# US 2014/0154689 A1 · Analysis of circulating tumor cells as diagnostic and predictive biomarkers for metastatic cancers

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- Number: US 2014/0154689 A1
- Document ID: US20140154689A1
- Status: Published application
- Kind: A1
- Application number: 14/098,112
- Filed: 2013-12-05
- Published: 2014-06-05
- Earliest claimed: 2012-12-05
- Inventors: Tim Hui-Ming Huang; Chun-Liang Chen; Joseph Liu; Chiou-Miin Wang; Devalingam Mahalingam; Pawel Osmulski
- Web page: https://patentgrep.com/patent/US20140154689A1

## CPC

- G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/158: Expression markers
- G01N 2333/70589: CD45
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method of analyzing circulating tumor cells includes separating circulating tumor cells from a subject's blood sample by filtration; separating CD45-negative cells from any retained blood cells; and determining the EMT-related gene expression profiles, nanomechanical and/or nanochemical properties of the collected CD45-negative CTC cells. Analysis of the collected circulating tumor cells enables the investigators to make a quick and accurate assessment of the metastatic behavior of the subject's circulating tumor cells for early castration-resistance and metastasis prediction in a clinical setting.

## Claims

### Claim 1

A method of analyzing circulating tumor cells comprising:
separating circulating tumor cells from a blood sample by filtration, wherein the circulating tumor cells represent at least a portion of cells retained by a filter of the filtration system;
separating CD45-negative cells from the retained cells on the filter; and
determining the nanomechanical and/or nanochemical properties of the collected CD45-negative cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein separating CD45-negative cells from the retained cells comprises:
staining the retained cells with an anti-CD45 antibody coupled to a visualization agent; and
physically separating the unstained circulating tumor cells from the stained cells.

### Claim 3 (depends on 2)

The method of claim 2, wherein physically separating the unstained circulating tumor cells from the stained cells comprises collecting the unstained circulating tumor cells using a micromanipulator and placing the collected unstained circulating tumor cells in a container.

### Claim 4 (depends on 1)

The method of claim 1, wherein determining the nanomechanical properties of the CD45-negative circulating tumor cells comprises determining the elasticity of the CD-45 negative circulating tumor cells.

### Claim 5 (depends on 4)

The method of claim 4, wherein determining the elasticity of the CD-45 negative circulating tumor cells comprises applying an atomic force microscope probe to at least one CD-45 negative circulating tumor cell and creating a cellular indentation mapping of the CD45-negative circulating tumor cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein determining the nanomechanical properties of the CD45-negative circulating tumor cells comprises determining the smoothness of the collected CD45-negative circulating tumor cells.

### Claim 7 (depends on 6)

The method of claim 6, wherein determining the smoothness of the collected CD45-negative circulating tumor cells comprises collecting one or more images of the collected CD45-negative circulating tumor cells and determining an average variance in height of the collected CD45-negative circulating tumor cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein determining the chemical features of the CD45-negative circulating tumor cells comprises determining the adhesiveness of the collected CD45-negative circulating tumor cells.

### Claim 9 (depends on 8)

The method of claim 8, wherein determining the adhesiveness of the collected CD45-negative circulating tumor cells comprises determining the force needed to pull a tip out of contact from a collected CD45-negative circulating tumor cell.

### Claim 10 (depends on 1)

The method of claim 1, further comprising lysing the collected CD45-negative circulating tumor cells and performing RT-PCR analysis of the lysed CD45-negative circulating tumor cells to determine the presence of metastatic specific genes.

### Claim 11

A method of identifying metastatic behavior of circulating tumor cells comprising:
separating circulating tumor cells from a blood sample by filtration, wherein the circulating tumor cells represent at least a portion of cells retained by a filter of the filtration system;
separating CD45-negative cells from the retained circulating tumor cells and blood cells;
determining the nanomechanical and/or nanochemical properties of the collected CD45-negative cells; and
determining the metastatic behavior of the collected CD45-negative cells based on the nanomechanical and/or nanochemical properties of the collected CD45-negative cells.

### Claim 12 (depends on 11)

The method of claim 11, wherein separating CD45-negative cells from the collected circulating tumor cells comprises:
staining the retained cells with an anti-CD45 antibody coupled to a visualization agent; and
physically separating the unstained circulating tumor cells from the stained cells.

### Claim 13 (depends on 12)

The method of claim 12, wherein physically separating the unstained circulating tumor cells from the stained cells comprises collecting the unstained circulating tumor cells using a micromanipulator and placing the collected unstained circulating tumor cells in a container.

### Claim 14 (depends on 11)

The method of claim 11, wherein determining the nanomechanical properties of the CD45-negative circulating tumor cells comprises determining the elasticity of the CD-45 negative circulating tumor cells.

### Claim 15 (depends on 14)

The method of claim 14, wherein determining the elasticity of the CD-45 negative circulating tumor cells comprises applying an atomic force microscope probe to at least one CD-45 negative circulating tumor cell and creating a cellular indentation mapping of the CD45-negative circulating tumor cell.

### Claim 16 (depends on 11)

The method of claim 11, wherein determining the nanomechanical properties of the CD45-negative circulating tumor cells comprises determining the smoothness of the collected CD45-negative circulating tumor cells.

### Claim 17 (depends on 16)

The method of claim 16, wherein determining the smoothness of the collected CD45-negative circulating tumor cells comprises collecting one or more images of the collected CD45-negative circulating tumor cells and determining an average variance in height of the collected CD45-negative circulating tumor cells.

### Claim 18 (depends on 11)

The method of claim 11, wherein determining the nanochemical properties of the CD45-negative circulating tumor cells comprises determining the adhesiveness of the collected CD45-negative circulating tumor cells.

### Claim 19 (depends on 18)

The method of claim 18, wherein determining the adhesiveness of the collected CD45-negative circulating tumor cells comprises determining the force needed to pull a tip out of contact from a collected CD45-negative circulating tumor cell.

### Claim 20 (depends on 11)

The method of claim 11, further comprising lysing the collected CD45-negative circulating tumor cells and performing microfluidic single-cell RT-PCR analysis of the lysed CD45-negative circulating tumor cells to determine the expression profile of metastatic specific genes.