Diagnostic Methods For Patient Specific Therapeutic Decision Making In Cancer Care

Abstract

The present invention relates to a 3-Dimensional (3D) tissue culture aggregate of cells derived from a neoplastic tissue sample, wherein 30% of total number cells are cells capable of interfering with re-aggregation. It also relates to a method of making such a 3D aggregate and a method for assessing the effectiveness of an anti-neoplasm treatment by measuring the effect of said treatment on the viability of a three dimensional (3D) neoplasm tissue culture aggregate.

Claims

1. A 3-Dimensional (3D) tissue culture aggregate of cells derived from a neoplastic tissue sample wherein 30% of total number cells are cells capable of interfering with re-aggregation; wherein said aggregate does not contain an artificial scaffold.

2. The 3D tissue culture aggregate of claim 1 wherein the cells capable of interfering with re-aggregation are lymphoid cells.

3. The 3D tissue culture aggregate of claim 1 wherein the cells capable of interfering with re-aggregation are CD45+.

4. A method for preparing a 3D tissue culture aggregate comprising: (a) Preparing an adjusted cell population from a neoplastic tissue sample by reducing the number of cells capable of interfering with re-aggregation to 30% of total number cells; and (b) Preparing a suspension culture comprising cells of said adjusted cell population, culture media and optionally fibroblasts; in the absence of an artificial scaffold.

5. The method of claim 4 wherein the number of fibroblasts in the initial suspension culture is 5-50% total number of cells.

6. The method of claim 4 wherein the number of cells from the adjusted cell population in the initial suspension culture is 2104 to 8106.

7. The method of claim 4 wherein the number of cells capable of interfering with re-aggregation is reduced by an immunological particle separation method or a cell sorting separation method.

8. The method of claim 4 wherein the extracellular matrix in the three dimensional (3D) neoplasm tissue culture aggregates is only produced by the cells themselves.

9. The method of claim 4, wherein the cells capable of interfering with re-aggregation are lymphoid cells.

10. The method of claim 4, wherein the cells capable of interfering with re-aggregation are CD45+.

11. The use of a 3D tissue culture aggregate of claim 1 to assess the effectiveness of an anti-neoplasm treatment.

12. A method for assessing the effectiveness of an anti-neoplasm treatment by measuring the effect of said treatment on the viability of a three dimensional (3D) neoplasm tissue culture aggregates.

13. The method of claim 12 wherein said 3D neoplasm tissue culture aggregates is a 3D tissue culture aggregate of claim 1.

14. The method of claim 12 wherein the viability of 3D neoplasm tissue culture aggregates is measured by using a cell viability assay.

15. The method of claim 12 further comprising determining the cellular composition of the 3D neoplasm tissue culture aggregates by cell surface marker analysis using flow cytometry.

16. The method of claim 12 further comprising assessing residual cancer stem cell drug sensitivity after a first anti-neoplastic agent treatment by (i) isolating neoplastic stem cells based on cell surface marker combinations; (ii) reaggregating isolated neoplastic stem cells into 3D tissue; and (iii) contacting the aggregated neoplastic stem cells with a second anti-neoplastic treatment, wherein said first antineoplastic treatment and said second antineoplastic treatment are different.

17. The 3D tissue culture aggregate of claim 2 wherein the cells capable of interfering with re-aggregation are CD45+.

18. The method of claim 5 wherein the number of cells from the adjusted cell population in the initial suspension culture is 2104 to 8106.

19. The method of claim 5 wherein the number of cells capable of interfering with re-aggregation is reduced by an immunological particle separation method or a cell sorting separation method.

20. The method of claim 6 wherein the number of cells capable of interfering with re-aggregation is reduced by an immunological particle separation method or a cell sorting separation method.

Description

[0148] The invention will now be described with reference to the following examples with refer to the following figures:

[0149] FIG. 1 shows Glioblastoma multiforme out-growth cultures.

[0150] FIG. 2 shows the results of flow cytometric analysis of glioblastoma multiforme.

[0151] FIG. 3 shows the response of Glioblastoma multiforme 3D aggregates after 72 hr incubation with various drugs.

[0152] FIG. 4 shows the response of Glioblastoma multiforme 3D aggregates after 24 hr incubation with different concentrations of BCNU.

[0153] FIG. 5 shows the results of flow cytometric analysis of adenocarcinoma pulmonis.

[0154] FIG. 6 shows the response of NSCLC Adenocarcinoma 3D aggregates after 72 hr incubation with different concentrations of monotherapies.

[0155] FIG. 7 shows the response of Testicular cancer 3D aggregates after 48 hr incubation with different concentrations and different combinations of drugs.

[0156] FIG. 8 shows the response of Malignant pleural fluid cells 3D aggregates after 48 hr incubation with different concentrations and different combinations of drugs

EXAMPLES

[0157] 1. Solid Tumour

[0158] 1.1 Primary Glioblastoma

[0159] Glioblastoma multiforme is one of the deadliest of neoplasms and continues to be regarded as incurable and universally fatal. This reputation seems well deserved, based on population-based outcome data from multiple centres over decades of investigation. Only a couple of percent of glioblastoma patients survive three years or longer, and five-year survival is still exceptionally rare.

[0160] Glioblastoma Multiforme Drug Sensitivity Analysis

[0161] Two, freshly resected, native samples reached the laboratory directly from the pathologist within 2 hours of surgery. The two samples were treated separately and were labelled as Glioblastoma 1 and Glioblastoma 2. The pathologist identified the macroscopically identical tumour samples as Glioblastoma 1 (Sample 1) being fully viable while Glioblastoma 2 (sample 2) as strongly necrotic. The samples were processed according to protocol and drug sensitivity tests were performed using the viable, Sample 1. Samples for DNA and RNA isolation were also stored at 80 C., leaving the opportunity open for additional sequencing or comparative gene expression studies. Traditional out-growth cultures were also prepared from Glioblastoma sample 1 showing the strong viability and proliferative ability of the cells (FIG. 1.).

[0162] Analysis Methods:

[0163] Toxicology assay: CellTiter-Glo 3D Cell Viability Assay (Promega). The CellTiter-Glo 3D Cell Viability Assay is a homogeneous, luminescent method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells.

[0164] Annexin: Annexin V is used as a non-quantitative probe to detect cells that express phosphatidylserine (PS) on their cell surface, an event found in apoptosis as well as other forms of cell death. The assay combines annexin V staining of PS and PE membrane events with the staining of DNA in the cell nucleus with propidium iodide (PI) or 7-Aminoactinomycin D (AAD-7), distinguishing viable cells from apoptotic cells and necrotic cells. Detection was performed by flow cytometry or a fluorescence microscope.

[0165] Cellular markers: GBM cancer stem cell markers: PROMININ-1/CD133, SSEA1/CD15, NESTIN, SOX2, BMI1, MUSASHI. Analysis is performed using flow cytometry and cytospin/tissue section staining and fluorescence microscopy (FIG. 2).

[0166] Drug Sensitivity Test

[0167] Aggregates were prepared in 96-well plates and cultures were incubated with the following agents: cisplatin, erlotinib, vinorelbine, and pemetrexed. 4 wells/treatment were tested, aggregates were cultured for 24, 48 or 72 h respectively, at 37 C. using the drugs in concentrations as: Cisplatin: 6 or 9 g/ml, Erlotinib (Tarceva): 100 nM or 1 M, Vinorelbine (Vinorelbine is a drug acting by a similar mechanism to Vincristine frequently used in neurooncology): 20 or 50 nM; Erbitux (Cetuximab): 4.8 mg/ml; BCNU (Carmustine): 0.3 mg/ml, 0.03 mg/ml, 0.003 mg/ml. Erlotinib (Tarceva) +Erbitux. Erlotinib similarly to Cetuximab is an EGFR inhibitor (the two drugs are frequently used clinically together). Following 24, 48 h or 72 h incubation, cells were labelled using Annexin V-PI and analyzed by flow cytometry or analysed by Promega CellTiter-Glo 3D Cell Viability Assay Kit (Luminescent) (ATP detection kit)(FIGS. 3 & 4).

TABLE-US-00001 Glioblastoma 1 Annexin Pl++ Pl+ ratio Late within Annexin+ apoptosis Non-viable % % SD 0.50 0.94 Cisplatin 9 g/m1 0.19 0.44 Erlotinib 1 M 0.32 0.62 Pemetrexed 1 M 0.19 0.37 Vinorelbine 20 nM 0.49 0.93 Vinorelbine 50 nM 0.05 0.09

[0168] The results clearly confirmed sensitivity of the glioblastoma cells to BCNU. The patient was treated with BCNU and the tumour was regressing within 2 weeks after the first administration of the drug.

[0169] 1.2 Non-Small Cell Lung Cancer

[0170] Eighty percent of all diagnosed lung cancers are non-small cell lung cancer. The 5-year survival rate of NSCLC varies from 73% in early detection (stage IA) to 3.7% at advanced metastatic disease. At early stages of NSCLC surgery and chemotherapy are still the choice of first line treatment, while in metastatic disease the focus is on chemotherapy.

[0171] NSCLC Drug Sensitivity Analysis

[0172] Freshly resected native lung carcinoma sample reached our laboratory within 24 h of surgery. Diagnosis was confirmed as NSCLC, adenocarcinoma pulmonis, (predominantly acinar, with a 30% lepidic component) pT1b N1. PN+, LI, R0.

[0173] Analysis Methods:

[0174] Toxicology assay: CellTiter-Glo 3D Cell Viability Assay (Promega). The CellTiter-Glo 3D Cell Viability Assay is a homogeneous, luminescent method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells.

[0175] Cellular markers: Analysis is performed using flow cytometry and cytospin/tissue section staining and fluorescence microscopy (FIG. 5).

[0176] Drug Sensitivity Test

[0177] Aggregates were prepared in 96-well plates and cultures were incubated with the following agents: cisplatin (6 or 9 g/ml), pemetrexed (50 nM and 100 nM), gemcitabine (50 nM and 1 M), docetaxel (1 nM and 10 nM), paclitaxel (1 nM and 10 nM) and their clinically applied combinations. 4 wells/treatment were tested, aggregates were cultured for 24, 48 h or 72 h at 37 C. Following incubation cells were analysed by Promega CellTiter-Glo 3D Cell Viability Assay Kit (Luminescent) (ATP detection kit) (FIG. 6).

[0178] The results clearly pointed out the cisplatin+gemcitabine combination as the most successful of chemotherapeutic combinations. The patient was treated with a Cisplatin+Gemcitabine combination and the disease has not been progressing.

[0179] 1.3 Testicular Cancer

[0180] Testicular cancer has one of the highest cure rates of all cancers with an average five-year survival rate of 95%. If the cancer has not spread outside the testicle, the 5-year survival is 99% while if it has grown into nearby structures or has spread to nearby lymph nodes, the rate is 96% and if it has spread to organs or lymph nodes away from the testicles, the 5-year survival is around 74%. Even for the relatively few cases in which cancer has spread widely, chemotherapy offers a cure rate of at least 80%.

[0181] Testicular Cancer Drug Sensitivity Analysis

[0182] Analysis Methods:

[0183] Toxicology assay: CellTiter-Glo 3D Cell Viability Assay (Promega). The CellTiter-Glo 3D Cell Viability Assay is a homogeneous, luminescent method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells.

[0184] Drug Sensitivity Test

[0185] Aggregates were prepared in 96-well plates and cultures were incubated with the following agents: cisplatin (6 or 9 g/ml), pemetrexed (50 nM and 100 nM), gemcitabine (50 nM and 1 M), docetaxel (1 nM and 10 nM), paclitaxel (1 nM and 10 nM) and their clinically applied combinations. 4 wells/treatment were tested, aggregates were cultured for 24, 48 h or 72 h at 37 C.

[0186] Following incubation cells were analysed by Promega CellTiter-Glo 3D Cell Viability Assay Kit (Luminescent) (ATP detection kit) (FIG. 7).

[0187] 2. Malignant Pleural Fluid

[0188] Malignant pleural effusion (MPE) usually presents in the disseminated and advanced stage of malignancy. Dyspnea is the debilitating symptom which needs palliation in these patients. By this stage of the disease there is no cure.

[0189] NSCLC Malignant Pleural Fluid Drug Sensitivity Analysis

[0190] Thoracentesis was performed on the patient who was presented with dyspnea and no prior diagnosis of neoplasm. Diagnosis was confirmed as NSCLC, adenocarcinoma, T4 Nx. M1.

[0191] Analysis Methods:

[0192] Toxicology assay: CellTiter-Glo 3D Cell Viability Assay (Promega). The CellTiter-Glo 3D Cell Viability Assay is a homogeneous, luminescent method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells.

[0193] Drug Sensitivity Test

[0194] Aggregates were prepared in 96-well plates and cultures were incubated with the following agents: cisplatin (6 or 9 g/ml), pemetrexed (50 nM and 100 nM), gemcitabine (50 nM and 1 M), docetaxel (1 nM and 10 nM), paclitaxel (1 nM and 10 nM) and their clinically applied combinations. 4 wells/treatment were tested, aggregates were cultured for 24, 48 h or 72 h at 37 C.

[0195] Following incubation cells were analysed by Promega CellTiter-Glo 3D Cell Viability Assay Kit (Luminescent) (ATP detection kit) (FIG. 8).