Methods and systems for analysis of single cells

Patent metadata

Field	Value
Publication number	US-9850483-B2
Application number	US-201113811065-A
Country	US
Kind code	B2
Filing date	Jul 19, 2011
Priority date	Jul 19, 2010
Publication date	Dec 26, 2017
Grant date	Dec 26, 2017

Abstract

Methods are provided for diagnosis and prognosis of disease by analyzing expression of a set of genes obtained from single cell analysis. Classification allows optimization of treatment, and determination of whether on whether to proceed with a specific therapy, and how to optimize dose, choice of treatment, and the like. Single cell analysis also provides for the identification and development of therapies which target mutations and/or pathways in disease-state cells.

First claim

What is claimed is:

1. A method of treating heterogeneous tumors in a human subject, comprising:
i. dissociating a tumor biopsy into individual cells, flowing the dissociated tumor biopsy through a microfluidic metamaterial to split the flow according to particle size and randomly partition the cells into a prefabricated microwell array;
ii. picking individual cells by expression of at least one cell surface marker from the microwell array and transferring each individual cell into a discrete location using an automated microfluidic system;
iii. performing transcriptome analysis on at least 50 genes of the individually selected cells; and
iv. identifying one or more therapeutic targets from transcriptome data obtained from the transcriptome analysis for treatment of said heterogeneous tumor.

2. The method of claim 1 , wherein the one or more therapeutic targets is a DNA or RNA methyltransferase, methyltransferase-like enzyme or a derivative thereof.

3. The method of claim 1 , wherein the one or more therapeutic targets is a histone lysine methyltransferase, histone arginine methyltransferase or a derivative thereof.

4. The method of claim 1 , wherein the one or more therapeutic targets is a histone demethylase or a derivative thereof.

5. The method of claim 1 , wherein the one or more therapeutic targets is a protein kinase or a derivative thereof.

6. A method of analyzing a heterogeneous tumor biopsy from a human subject or treatment of the tumor from the human subject, comprising:
i. dissociating the tumor biopsy into individual cells, flowing the dissociated tumor biopsy through a microfluidic metamaterial to split the flow according to particle size and randomly partition the cells into a prefabricated microwell array;
ii. picking individual cells by expression of at least one cell surface marker from the microwell array and transferring each individual cell into a discrete location using an automated microfluidic system;
iii. performing transcriptome analysis on at least 50 genes of the individually selected cells; and
iv. identifying one or more diagnostic markers present in the tumor biopsy from transcriptome data obtained from the transcriptome analysis for the detection of cancer and determining of the cancer stage or the effectiveness of treatment of the tumor from the human subject.

7. The method of claim 6 , wherein the one or more diagnostic markers is a DNA or RNA methyltransferase, methyltransferase-like enzyme or a derivative thereof.

8. The method of claim 6 , wherein the one or more diagnostic markers is a histone lysine methyltransferase, histone arginine methyltransferase or a derivative thereof.

9. The method of claim 6 , wherein the one or more diagnostic markers is a histone demethylase or a derivative thereof.

10. The method of claim 6 , wherein the one or more diagnostic markers is a protein kinase or a derivative thereof.

11. The method of claim 1 , wherein the at least 50 genes comprise one or more of STAT3, MEIS1, CAV1, GAS1, MAP4K4 (kinase) MYLK (kinase), PTK2 (kinase), DAPK1 (kinase), LATS (kinase), FOSL2, AKT3 (kinase), PTPRC (tyrosine phosphatase), MAFF (oncogene), RRAS2, NFKB, ROBO1, IL6ST, CR1M1, PLS3, CXCL14, ETS1, ETS2, CD47, RGS4, CAV2, MAF, WT1, SNAI2, MEIS2, ID4, FOXC1, GSS, GCLC, GCLM, GPX1, GPX4, GPX7, SLPI, PRNP, SOD1, SOD2, SOD3, CAT, NFKB1, FOXO1, FOXO3A, FOXO4, KRT19, CHI311, TERT, HIF1A, EPAS1, HPRT, and ACTB.

Assignees

• Clarke Michael F
• Quake Stephen R
• Dalerba Piero D
• Liu Huiping
• Leyrat Anne A
• Kalisky Tomer
• Diehn Maximilian
• Rothenberg Michael
• Wang Jianbin
• Lobo Neethan
• Univ Leland Stanford Junior

Inventors

• Clarke Michael F
• Quake Stephen R
• Dalerba Piero D
• Liu Huiping
• Leyrat Anne A
• Kalisky Tomer
• Diehn Maximilian
• Rothenberg Michael
• Wang Jianbin
• Lobo Neethan

Classifications

• C12Q2600/158

  Expression markers · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12N15/1072 Primary

  Differential gene expression library synthesis, e.g. subtracted libraries, differential screening · CPC title