Patent Application
20200190470
The present invention relates to temozolomide resistant glioblastoma cells lines. More particularly, the present invention relates to a method for characterizing temozolomide resistant cells derived from glioblastoma cells lines in patients diagnosed with glioblastoma and undergoing treatment with temozolomide and/or in patients on the part to recovery to avoid and treat relapse.
Glioblastoma multiforme (GBM) is a devastating form of brain cancer with a dismal median survival time, a high level of resistance to current therapy and common recurrence after treatment. The current standard therapy for GBM includes maximum debulking surgery, radiation and treatment with the monofunctional alkylating agent temozolomide (TMZ). Temozolomide (TMZ), an anti-cancer prodrug of Temodar® is an oral alkylating agent that is used in the treatment of glioblastoma multiforme (GBM) and astrocytomas. Temozolomide is an imidazotetrazine that has increased the prognosis of highly aggressive GBM, however, at least 50% of TMZ treated patients do not respond to TMZ.
Primarily, this is due to the over-expression of O6-methylguanine methyltransferase (MGMT) and/or lack of a DNA repair pathway in GBM cells. In the absence of MGMT repair, O6-methylguanine is suggested to initiate a futile cycle of mismatch repair (MMR) or alternately to trigger ataxia telangiectasia and Rad3 related protein kinase (ATR) activation through the action of several MMR proteins, leading to apoptosis and cell death.
Much of the resistance to TMZ observed clinically is due to high expression of MGMT (and subsequent repair of the lesion) or loss of MMR (therefore preventing the initiation of apoptotic signalling). Additionally, almost all patients eventually recur with the disease and the large majority of recurrent tumors are resistant to chemotherapy. There are currently few alternate treatment options for patients with TMZ resistant tumors and adjuvant chemotherapy options are an area of intense research.
The following GBM cells were reported consistently to be TMZ resistant: LN-18, T98G, UL38, CCF-STTG1 and U343-MG GBM cells.
The existence of small minority populations with differential histology and dye efflux properties within cancer cell lines has been known for decades; however, the underlying biochemical physiology of how this shapes functional drug response, susceptibility and resistance is still incompletely understood.
There is a need in the an to identify and characterize temozolomide resistant cancer cells in terms of drug efflux, growth/proliferation, nutrient preferences and metabolite profiling to drug dose response, thereby providing a tool for diagnosis and treatment of temozolomide resistance of glioblastoma cancer cells and its relapse.
It is an object of the present invention to identify and characterize temozolomide resistant cancer cells derived from a parental glioblastoma cell line, so that the physiological contributors to drug resistance in cancer cells can be identified.
It is another object of the present invention to provide a method for screening temozolomide resistant cancer cells in patients diagnosed with glioblastoma and undergoing treatment with temozolomide and/or in patients on the part to recovery to avoid relapse.
In an aspect, the present invention provides temozolomide resistant neurospheroidal cells (NSP) derived from glioblastoma cells having the following characteristics:
In another aspect, the present invention provides a method for screening temozolomide resistant neurospheroidal (NSP) cells from glioblastoma cell lines, the said method comprising;
In yet another aspect, the present invention provides a kit for identification and characterization of temozolomide resistant cells lines in patients diagnosed with glioblastoma and undergoing treatment with temozolomide, the said kit comprising temozolomide resistance cell lines derived from U87MG (Grade-IV GBM cell line) along with instructions crucial for identifying, screening and characterization of the said cells, wherein the said instructions comprise;
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
In a preferred embodiment, the present invention provides temozolomide resistant neurospheroidal (NSP) cells derived from glioblastoma cell lines having the following characteristics:
In accordance with the aforesaid embodiment, the present invention provides neurospheres (NSP) as small spheroidal cells forming floating aggregates (
As regards in-vitro growth differentiation of the NSP cells vis-à-vis the parental glioblastoma cell line, the present invention provides the NSP growth rate is 30% lower than the parental glioblastoma cell line and thus the maximum population density achieved at the end of 216 h was higher for U87MG as compared to NSP.
As regards Temozolomide dose response curves, the present invention provides a higher IC50 for NSP (1039 mM) indicating a potential lower efficacy of the drug on these cells (
As regards the IC50 values of NSP cells differentiated into glial cell types, these differentiated cells of NSP showed a TMZ dose response in between that of NSP and U87MG, exhibiting an IC50 value of 817.5 mM and growth rate of 0.01634 hr−1.
As regards, the metabolite profiling in NSP cells and the parental cell line, it was observed in the present invention that NSP only secreted alanine and glutamate. Glycine and glutamate were secreted initially in fast dividing cells and then taken-up later suggesting a metabolic functional role in NSP.
Another feature distinct from the parental cell line, i.e. U87MG was that NSP however seems to show maximum uptake of these amino acids after a 24 h lag. The utilization of the amino acids Tyr, Trp, Val, Thr, Ser, Pro, Met, Phe, Lys, Leu/Ile, His in NSP cells is somewhere between 120 and 168 h that continued until growth was monitored to 216 h (
In another preferred embodiment, the present invention provides a method for screening temozolomide resistant neurospheroidal (NSP) cells from glioblastoma cell lines, the said method comprising;
In a further embodiment, the present invention provides Temozolomide to be potentially efficacious in controlling the NSP growth but only at concentrations that are 40% higher than the IC50 value ranging from 1000 μM to 1100 μM.
In another embodiment, the present invention provides that TMZ resistance is in part due to the effect of specific receptor tyrosine kinases and substrate ABC transporters that rewire metabolism through preferential utilization.
A higher level of cytokine STAT3 in NSP potentially turns on the MYC transcriptional program and induces preferential consumption of glutamine. The simultaneous negative regulation of ACL by AKT1 potentially lowers the acetyl CoA pool and hence the acetate available for histone acetylation. A reduced aerobic glycolytic flux reflective of higher NAD levels may control SIRTUIN family transcriptional repressors and silence chromatin via decreasing histone acetylation. This partially explains differential tryptophan metabolism (growth limiting U87MG) and suggests a role for picolinate carboxylase in the balance between NAD derived nucleotide synthesis and acetyl-CoA driven acetylation. Higher levels of AKG (TCA pool) also indicate epigenetic connections to the Jumonji family of histone de-methylases that could prevent methylation of histones. This may explain the varied dose response relations and increased IC50 for TMZ. The dependency on glucose and pyruvate, and not glutamine for growth/respiration also suggests normal levels of methylation in U87MG. Similar MGMT mRNA levels suggest functional DNA repair in the absence of TMZ. The role of AKG in demethylation of DNA and its effects on drug sensitivity/resistance needs further testing.
In a further embodiment, the present invention provides a kit for identification and characterization of temozolomide resistant cells lines in patients diagnosed with glioblastoma and undergoing treatment with temozolomide, the said kit comprising temozolomide resistance cell lines derived from U87MG (Grade-IV GBM cell line) along with instructions crucial for screening and characterization the said cells, wherein the said instructions comprise;
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
An authenticated U87MG cell line (HTB-14; Human Glioblastoma Multiforme from ATCC;) was cultured in conditions as per ATCC guidelines. Neurospheres (NSP) were maintained in neurobasal medium supplemented with B27 supplement, 0.2 mg/mL each of epidermal growth factor, EGF and basic fibroblast growth factor, (bFGF). NSP were cultured as floating spheres in low attachment T-75 flasks or 6 well/24 well plates (Nunc™). All chemicals and labware was purchased from ThermoFisher Scientific™.
The glioblastoma cell line U87MG contained a sub-population (0.1%) of Hoechst-effluxing cells. The sub-population (NSP) was confirmed with Verapamil, an ABC transporter L-type calcium channel blocker and inhibitor of dye efflux. The separated populations were tested for morphological and phenotypic heterogeneity and temozolomide dose response.
Hoechst 33342 stain (1 mg/mL) was used for all fluorescence studies on EVOS® FLoid® cell imaging system. The subpopulation sorting assay as previously described was performed for FACS with cells at 70-80% confluency using BD FACSAria III (BD biosciences Pvt. Ltd) and analysed using BD FACSDiva™ software v6.1.3. Cells were captured in a Hoechst Blue versus Hoechst Red dot plot in the presence and absence of Verapamil.
Under bright field microscopy, cultures of separated U87MG showed glial cell characteristics with epithelial cell morphology (
The multi-step gating strategy based on differential fluorescence profiles was critical for characterizing and sorting the subpopulation from the main population. The flow-cytometric profile based on the forward scatter (FSC, indicative of cell size) and side scatter (SSC, indicative of cell granularity) allowed distinguishing viable cells from cell debris. NSP cells were recognized as a dim tail extending first on the left side towards the lower “Hoechst Blue” signal (
Growth/proliferation of the cells (Parental U87MG, U87MG and NSP) was monitored via cell counts over a period of 216 h (9 days). The initial seeding (No) was ˜10000 cells per well. All cells were harvested every 24 h and counted using hemocytometer using trypan blue dye exclusion assay. NSP was trypsinized before counting. Growth curves were graphed and data fitted with Gompertz function (GraphPad Software, San Diego Calif. USA).
Growth profiles (
N(t)=No exp(ln(N(t)/No)[1−exp(−kt)]
where No defines the initial seeding density of the cells, Nt is the number of cells at time t, and k is the maximum specific growth constant (Table 1).
higher than that of NSP
to
and
for U87MG and NSP respectively.
growth
10
10
10
indicates data missing or illegible when filed
The doubling times calculated from the growth rates (Table 1) were 35.12 and 47.05 h for U87MG and NSP respectively. The fitted Gompertz function (
For dose-response experiments, four replicates at ˜20,000 cells per well were plated in 96-well Nunc™ tissue culture plates in full growth medium for 24 h, treated with different doses of TMZ in serial dilutions (0.05Me5M) followed by cell viability tests using the MTT assay. Appropriate cell controls (without TMZ treatment) were used to calculate IC50.
The varied dose response and growth inhibition patterns of U87MG and NSP are observed in the steepness of the dose response curve for TMZ (Temodar) (
However, a HS value of near 0 for NSP indicates a potentially higher efficacy, at least in culture. AUC combines potency and efficacy of a drug into a single parameter. AUC values were compared across U87MG and NSP exposed to the same range of TMZ concentrations indicate higher impact of the drug on U87MG. Temozolomide is potentially also shown to be efficacious in controlling the NSP growth but only at concentrations that are 40% higher (indicated by fold resistance ratio 1.41).
NSP were grown in DMEM followed by addition of 1% N2 supplement. Growth factors, bFGF and EGF reconstituted in 0.1% BSA solution (100 mg/mL) were used. An increased B27 (2%) supplemented after 3 days drove the differentiation. The differentiated cells (NDx) were cultured separately using the same method of culture of U87MG cells. The ability of NSP to undergo differentiation and form specialized cell types was confirmed in an assay with specific growth factors and supplements. The differentiated cells (NDx) exhibited glial morphology and were adherent in contrast to the NSP (spheroidal) population (
Samples harvested during growth every 24 h over a period of 9 days were used for metabolic profiling performed in Accela 1250 ultra-performance liquid chromatography (UPLC) in tandem with Thermo Q-exactive high resolution mass spectrometer (HRMS) using heated electrospray ionization (HESI) interface. Parameters of LC-HRMS run and sample extraction protocols and standard preparation are provided. Accurate mass-extracted ion chromatograms (AM-XIC) of various metabolites were generated followed by peak confirmation by MS/MS spectral peak matching. Confirmed metabolites were quantified using internal standard normalized linear regression models. The raw and processed data of MS/MS confirmations, concentration ranges and regression fits are provided.
Quantitative exo-metabolite profiling identifies differential dynamics of nutrient uptake in the NSP population. LC-MS/MS was used to monitor the differential nutrient uptake of glucose and amino acids required for growth (
The maximum uptake rates of these amino acids in U87MG being highest in the first 24 h decreasing several fold by the end of 96 h. NSP however seems to show maximum uptake of these amino acids after a 24 h lag. The utilization patterns indicate a linear decrease of these nutrients for U87MG while during NSP growth, an exponential decrease of the same nutrients was observed with a 48 h lag corresponding to the end of the first doubling (
NSP however only secreted alanine and glutamate. Glycine and glutamate were secreted initially in fast dividing cells and then taken-up later suggesting a metabolic functional role in NSP. Several amino acids (Cys, Leu/Ile, Lys, Gly, Met, Phy, Ser, Thr, Tyr and Val) were utilized linearly in the first 48 h of growth by U87MG. The same was observed in NSP only after the first doubling (
The differential mRNA abundances of signalling pathway controllers like the receptor tyrosine kinases may shape metabolic dynamics while modulation in histone deacetylases and methyl transferases potentially contribute to changes observed in TMZ dose response relation (increasing IC50 values and decreasing sensitivity). It is well known that p53 and PTEN play a pivotal role in tumor suppression and both have less mRNA in U87MG with respect to NSP (
Higher mRNA of AKT1 in U87MG with respect to NSP is evident in the differential glucose and amino acid uptake patterns. IDH1/IDH2 has higher mRNA in NSP in comparison with U87MG indicating potential reprogramming of the pathways around AKG that could eventually cause downstream effects on the acetylation profiles. Increased PIK3CA (5 fold change) in NSP could potentially dictate changes involving phosphorylation governing differential nutrient uptake. VEGFA and MDM2 also have higher mRNA expressed in NSP compared to U87MG. Lower CDK4 mRNA abundances as compared to U87MG indicated a potential cell cycle arrest that needs to be overcome for NSP to proliferate. Lowered H3F3A mRNA in NSP indicates potential changes in histone acetylation patterns. The gene expression of 19 out of the 44 ATP-binding cassette (ABC) transporters profiled, varied between NSP and U87MG (
Biolog Phenotype Micro-Arrays™ PM-M1-M2 from Biolog, Inc. USA (www.biolog.com) were tested using protocols suggested by the manufacturer. All plates were incubated at 37° C. in CO2 incubator and absorbance monitored at 590 nm at regular time intervals to calculate respirations rates. Phenotypic plasticity defined by nutrient preferences for growth and respiration. The cellular energetics and pathways involved in the metabolism of U87MG and NSP during the nutrient restricted state (only one carbon or nitrogen source) were addressed using Biolog Phenotype MicroArrays™ plates (PM-M and PM-M2). The varied metabolic fingerprints of respiration and growth on 73 carbon and nitrogen sources were analyzed using Euclidean clustering (
The present invention provides integrative paradigms across molecular hierarchies in the cell which helps to design combinatorial treatments using metabolite supplements to overcome drug resistance.
