Methods for identifying, purifying and enriching immature or stem cancer-initiating cells from tumors and use thereof
AssigneesHopitaux Universitaires De Geneve, Universite De Geneve
InventorsIvan Radovanovic, Virginie Clement
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Abstract
The present invention is related to methods for the preparation of cell compositions, isolated compositions obtainable therefrom, related isolated cell compositions, kits and use thereof. More specifically, the present invention provides a method for identifying, purifying and enriching immature or stem cancer-initiating cells in a 5 sample. The cell compositions, related methods and uses according to the present invention are useful in the treatment of cancers and/or the detection of enriching immature or stem cancer-initiating cells, notably cancers of the central and peripheral nervous system, metastasis to the brain.
Claims30
A method for the preparation of a cell composition, comprising the steps of:
- (a) Providing a tumor cell sample;
- (b) Optionally culturing the cells provided in (a) in a culture medium;
- (c) Isolating in a sub-sample the cells which present autofluorescence emission detected in the FL1 channel upon laser beam excitation at a wavelength of or about 488 nm by fluorescence activated cell sorting, from the cells provided under step (a) or (b);
- (d) Isolating in another sub-sample by fluorescence activated cell sorting, the cells which are not fluorescent under step (c) and which present a slight positive shift in the fluorescence detected in the FL3 and/or FL4 channel;
- (e) Excluding dead cells from each of the isolated cell sub-sample obtained under steps (c) and (d);
- (f) Pooling the cell sub-sample obtained under step (c) after treatment under step (e);
- (g) Pooling the cell sub-sample obtained under step (d) after treatment under step (e).
An isolated cell composition comprising living cells which present an autofluorescence emission detected in the FL1 channel upon laser beam excitation at or about 488 nm by fluorescence activated cell sorting.
An isolated cell composition comprising living cells which do not present an autofluorescence emission detected in the FL1 channel upon laser beam excitation at or about 488 nm and present a slight positive shift in the fluorescence detected in the FL3 and/or FL4 channel by fluorescence activated cell sorting.
A method of detecting the presence of stem or immature cancer cells in a cell sample, comprising the following steps:
- (a) Providing a cell sample;
- (b) Optionally culturing the cells provided in (a) in a culture medium;
- (c) Detecting cells by fluorescence activated cell sorting cell which present autofluorescence emission in the FL1 channel upon laser beam excitation at a wavelength of or about 488 nm from cells provided under step (a) or (b);
- (d) Detecting cells by fluorescence activated cell sorting which do not present autofluorescence emission in the FL1 channel under step (c) and present a slight positive shift in the cell fluorescence emission in the FL3 and/or FL4 channel upon laser beam excitation of cells provided under step (a) or (b);
- (e) Determining the ratio of fluorescence signals obtained under step (d) and obtained under step (c).
A method of detecting the recurrence of cancer stem cells in a cancer stem cell sample, comprising the following steps:
- (a) Providing a cancer stem cell sample;
- (b) Treating the cancer stem cell sample provided under (a) with an agent;
- (c) Incubating the treated stem cell sample in a stem cell culture medium for an incubation period without treatment;
- (d) Selecting the viable cell population from the stem cell sample incubated under step (c);
- (e) Measuring the mean level of autofluorescence on the viable cell population isolated under step (d) by detecting, by fluorescence activated cell sorting, cells presenting autofluorescence emission in the FL1 channel upon laser beam excitation at a wavelength of or about 488 nm;
- (f) Isolating cells by fluorescence activated cell sorting which have a high FSC and low/middle SSC morphology and present autofluorescence emission in the FL1 channel upon laser beam excitation at a wavelength of or about 488 nm of the viable cell population isolated under step (d);
- (g) Isolating cells by fluorescence activated cell sorting which have a low/middle FSC and middle/high SSC morphology, do not present autofluorescence emission in the FL1 channel under step (d) and present a slight positive shift in the cell fluorescence emission in the FL3 and/or FL4 channel upon laser beam excitation of the viable cell population isolated under step (d);
- (h) Calculating the percentage of autofluorescent viable cells by comparing the mean level of autofluorescence in the cancer stem cell sample provided under step (a) and the mean level of autofluorescence measured under step (e);
- (i) Calculating the percentage of the cell death by comparing the number of initial cells present in the cancer stem cell sample provided under step (a) and the resulting viable cell population isolated under step (d);
- (j) Calculating the percentage of viable FL1+ cells by comparing the number of initial FL1+ cells present in the cancer stem cell sample provided under step (a) and the resulting viable FL1+ cell population isolated under step (f);
- (k) Calculating the percentage of viable FL10 cells by comparing the number of initial FL10 cells present in the cancer stem cell sample provided under step (a) and the resulting viable FL10 cell population isolated under step (g);
- (l) Detecting spherogenicity of the cell populations detected under steps (f) and (g).
- (m) Determining the activity of the agent through its ability to inhibit cancer stem cells recurrence.
Dependencies are inferred from the claim text.