Methods and systems for analysis of single cells

• US 9,850,483 B2
• Filed: 07/19/2011
• Issued: 12/26/2017
• Est. Priority Date: 07/19/2010
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• 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
• Primary Examiner(s): Ghafoorian, Reza
• Application Number:
  US13/811,065
• Publication Number:
  US 20130225435A1
• Time in Patent Office: 2,352 Days
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 Expression markers

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.

Claims

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.

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.

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.