Single cell gene expression for diagnosis, prognosis and identification of drug targets

• US 20100255471A1
• Filed: 01/20/2010
• Published: 10/07/2010
• Est. Priority Date: 01/20/2009
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Stanford University
• Inventors: Kalisky, Tomer, Dalerba, Piero D., Leyrat, Anne, Liu, Huiping, Diehn, Maximilian, Quake, Stephen R., Clarke, Michael F.
• Granted Patent: US 9,329,170 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

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 identifying different cell populations in a heterogeneous solid tumor sample, comprising:

randomly partitioning individual cells from said tumor into discrete locations;

performing transcriptome analysis on a plurality of genes of the individually partitioned cells in the discrete locations; and

performing clustering analysis to identify one or more different cell populations.

2. The method of claim 1, wherein said individual cells are not enriched prior to said partitioning.

3. The method of claim 1, wherein said transcriptome analysis is performed on at least 1000 individual cells simultaneously.

4. The method of claim 1, wherein said transcriptome analysis is performed using nucleic acid analysis.

5. The method of claim 1, wherein said discrete locations are on a planar substrate.

6. The method of claim 1, wherein said random partitioning is performed in a microfluidic system.

7. The method of claim 1, wherein said transcriptome analysis comprises analyzing expressed RNA, non-expressed RNA, or both.

8. The method of claim 1, wherein said transcriptome analysis is whole transcriptome analysis.

9. The method of claim 1, wherein said transcriptome analysis comprises amplifying RNA using a single set of primer pairs.

10. The method of claim 9, wherein said primer pairs are not nested primers.

11. The method of claim 1, wherein said transcriptome analysis is performed simultaneously or substantially in real time on all or a subset of said individual cells.

12. The method of claim 1, wherein said one or more cell populations are normal stem cells, normal progenitor cells, normal mature cells, inflammatory cells, cancer cells, cancer stem cells or non-tumorigenic stem cells.

13. A method of analyzing a heterogeneous tumor biopsy from a subject, comprising:

randomly partitioning cells from the biopsy into discrete locations;

performing transcriptome analysis on at least 50 genes of the individually partitioned cells; and

using transcriptome data to identify one or more characteristic of said tumor.

14. The method of claim 13, wherein the performing step is done without prior enrichment of a cell type.

15. The method of claim 13, wherein the characteristic is the presence, absence, or number of cancer cells.

16. The method of claim 13, wherein the characteristic is the presence, absence or number of stem cells, early progenitor cells, initial differentiated progenitor cells, late differentiated progenitor cells, or mature cells.

17. The method of claim 13, wherein the characteristic is effectiveness of a therapeutic agent in eliminating one or more of the cells.

18. The method of claim 13, further comprising using said characteristic to diagnose said subject with cancer or a cancer stage.

19. The method of claim 13, wherein the characteristic is activity of a signaling pathway.

20. The method of claim 19, wherein said signaling pathway is specific to a cancer stem cell, a differentiated cancer cell, a mature cancer cell, or combination thereof

21. A method of identifying a signaling pathway utilized by a disease-state cell, comprising:

randomly partitioning cells from a heterogeneous sample;

performing transcriptome analysis on said partitioned cells;

using transcriptome analysis to identify at least one disease-state cell;

comparing said transcriptome analysis of said at least one disease-state cell to transcriptome of;

a) a non-disease state cell;

b) a different disease-state cell; and

c) a disease-state stem cell; and

identifying a signaling pathway that is expressed in (i) the disease-state cell, (ii) the disease-state stem cell, and (iii) optionally in the different disease-state cell, but not in a non-disease-state cell, thereby identifying a signaling pathway utilized by a disease-state cell.

22. The method of claim 21, wherein said disease state is cancer, ulcerative colitis or inflammatory bowel disease.

23. The method of claim 21, wherein said signaling pathway is required for survival of said disease state cell.

26. The method of claim 21, wherein said transcriptome analysis comprises analyzing expressed RNA, non-expressed RNA, or both.

27. The method of claim 21, wherein said transcriptome analysis is whole transcriptome analysis.

28. The method of claim 21, wherein said disease state cell is a breast cancer stem cell.

24. A method for diagnosing a subject with a condition comprising, randomly partitioning cells from a heterogeneous sample;

performing a first transcriptome analysis on partitioned cells;

using transcriptome analysis to identify at least one disease-state cell by comparing said first transcriptome analysis from said at least one disease-state cell to a second transcriptome analysis from a non-disease state cell, thereby diagnosing the presence or absence of a condition associated with said disease state cell in said subject.

25. The method of claim 24, wherein said disease state is breast cancer, colon cancer, ulcerative colitis or inflammatory bowel disease.

29. A method for screening for a therapeutic agent comprising:

exposing a first subject with disease-state cells to one or more test agents;

obtaining a heterogeneous tumor biopsy from the subject from a region of interest;

performing transcriptome analysis on at least one individual cell from said heterogeneous tumor biopsy,

wherein said biopsy comprises one or more disease state cells; and

comparing said transcriptome analysis to a transcriptome derived from either;

(i) a second subject without the disease-state cells;

or

(ii) the first subject prior to said exposing step; and

identifying an agent that affect transcriptome of cells from the test area to be more like those of the second subject or the first subject prior to exposure.

30. A method of determining the potential effectiveness of a therapeutic agent against a disease, comprising:

separating a first population of disease-state cells into individual locations, wherein said individual locations comprise an individual cell;

determining the expression level of at least one nucleic acid or protein from at least one of said individual cells, thereby producing a disease-state expression signature;

exposing a second population of disease state cells to an agent;

separating said second population of disease-state cells into individual locations, wherein said individual locations comprise an individual cell;

determining the expression level of at least one nucleic acid or protein from at least one of said individual cells from said second population; and

comparing said expression level from said individual cell from said second population to said disease-state expression signature, thereby determining the effectiveness of said agent against said disease.

31. The method of claim 30, wherein said exposing step is performed in vivo.

32. The method of claim 30, wherein said first population and said second population are isolated from a subject.

33. The method of claim 32, wherein said subject is a human.

34. The method of claim 30, wherein said disease is cancer, ulcerative colitis or inflammatory bowel disease.

35. The method of claim 30, wherein said nucleic acid or said protein is a cancer stem cell marker.

36. The method of claim 30, wherein said expression level is mRNA expression level.

37. The method of claim 33, wherein determining said mRNA expression level comprises detection of expression or lack of expression of 10 or more nucleic acids.

38. The method of claim 30, wherein said expression level is protein expression level.

39. The method of claim 30, wherein said separating steps comprise exposing said population of cells to an antibody that specifically binds a protein present on said individual cells.

40. A method of determining likelihood of a response by a subject to a therapeutic agent, comprising:

separating a population of cells from a subject into individual locations, wherein said individual locations comprise an individual cell and wherein at least one of said individual cells is a disease-state cell;

determining the expression level of at least one nucleic acid or protein from at least one of said disease-state individual cells, wherein said nucleic acid or protein is a target of a therapeutic agent; and

determining likelihood of a response by a subject based on the expression level of said at least one nucleic acid or protein.

41. The method of claim 40, wherein said expression level is mRNA expression level.

42. The method of claim 41, wherein determining said mRNA expression level comprises detection of expression or lack of expression of 10 or more nucleic acids.

43. The method of claim 42, wherein said expression level is protein expression level.

44. The method of claim 42, wherein said separating step comprises exposing said population of cells to an antibody that specifically binds a protein present on said individual cells.

45. The method of claim 40, wherein said therapeutic agent is an anti-cancer agent.