Method of restoring sensitivity to TTField in TTField-resistant cancer cells using PTGER3 inhibitors
阅读说明：本技术 用PTGER3抑制剂恢复TTField抗性癌细胞中对TTField的敏感性的方法 (Method of restoring sensitivity to TTField in TTField-resistant cancer cells using PTGER3 inhibitors ) 是由 D·陈 S·B·乐 于 2020-03-27 设计创作，主要内容包括：提供了方法,所述方法通过向受试者推荐或开PTGER3抑制剂处方并向癌细胞施加交变电场来降低癌细胞的活力、防止受试者的癌细胞发展对TTField的抗性和恢复癌细胞对TTField的敏感性。在一些情况下,可以用一种或多种PTGER3抑制剂(例如,NSAID、cox2抑制剂)恢复癌细胞对TTField的敏感性。(Methods are provided for reducing the viability, preventing the development of resistance to and restoring sensitivity to TTField of cancer cells in a subject by recommending or prescribing a PTGER3 inhibitor to the subject and applying an alternating electric field to the cancer cells. In some cases, sensitivity of cancer cells to TTField can be restored with one or more PTGER3 inhibitors (e.g., NSAIDs, cox2 inhibitors).)
1. A method of reducing the viability of a TTField-resistant cancer cell in a subject, the method comprising:
recommending to the subject administration of a prostaglandin E receptor 3 (PTGER3) inhibitor; and
applying an alternating electric field to the cancer cells of the subject, the frequency of the alternating electric field being between 100-500 kHz.
2. The method of claim 1, wherein the frequency of the alternating electric field is between 100 and 300 kHz.
3. The method of claim 1, wherein the PTGER3 inhibitor is selected from one or more of an NSAID, a cox2 inhibitor, L798,106, and DG 041.
4. The method of claim 1, wherein the PTGER3 inhibitor is selected from aspirin and ibuprofen.
5. The method of claim 4, wherein the recommended concentration of the PTGER3 inhibitor in the subject is about 1-500 nanomolar for L798,106, or 0.1-2 millimolar for aspirin, or 0.5-50 nanomolar for DG041, or 1-500 nanomolar for celecoxib.
6. The method of claim 5, wherein the recommended concentration of the PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks.
7. The method of claim 1, wherein the cancer cell is selected from the group consisting of glioblastoma, lung cancer, pancreatic cancer, mesothelioma, ovarian cancer, and breast cancer cells.
8. A method of preventing cancer cells in a subject from developing resistance to an alternating electric field, the method comprising:
recommending to the subject administration of a prostaglandin E receptor 3 inhibitor; and
applying an alternating electric field to the cancer cells of the subject, the frequency of the alternating electric field being between 100-500 kHz.
9. The method of claim 8, wherein the frequency of the alternating electric field is between 100 and 300 kHz.
10. The method of claim 8, wherein the PTGER3 inhibitor is selected from the group consisting of NSAIDs, cox2 inhibitors, L798,106, and DG 041.
11. The method of claim 10, wherein the PTGER3 inhibitor is selected from aspirin and ibuprofen.
12. The method of claim 11, wherein the recommended concentration of the PTGER3 inhibitor in the subject is about 1-500 nanomolar for L798,106, or 0.1-2 millimolar for aspirin, or 0.5-50 nanomolar for DG041, or 1-500 nanomolar for celecoxib.
13. The method of claim 12, wherein the recommended concentration of the PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks.
14. The method of claim 8, wherein the cancer cell is selected from the group consisting of glioblastoma, lung cancer, pancreatic cancer, mesothelioma, ovarian cancer, and breast cancer cells.
15. A method of restoring sensitivity to TTField in TTField-resistant cancer cells of a subject, the method comprising recommending to the subject administration of a PTGER3 inhibitor, wherein sensitivity to TTField in the TTField-resistant cancer cells of the subject is substantially restored.
16. The method of claim 15, wherein the PTGER3 inhibitor is selected from the group consisting of NSAIDs, cox2 inhibitors, L798,106, and DG 041.
17. The method of claim 16, wherein the PTGER3 inhibitor is selected from aspirin and ibuprofen.
18. The method of claim 17, wherein the recommended concentration of the PTGER3 inhibitor in the subject after administration of the PTGER3 inhibitor to the subject is about 1-500 nanomolar for L798,106, 0.1-2 millimolar for aspirin, or 0.5-50 nanomolar for DG041, or 1-500 nanomolar for celecoxib.
19. The method of claim 18, wherein the recommended concentration of the PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks following administration of the PTGER3 inhibitor to the subject.
20. The method of claim 15, wherein the cancer cell is selected from the group consisting of glioblastoma, lung cancer, pancreatic cancer, mesothelioma, ovarian cancer, and breast cancer cells.
21. A method of reducing the viability of a TTField-resistant cancer cell in a subject, the method comprising:
prescribing a PTGER3 inhibitor to the subject; and
applying an alternating electric field to the cancer cell, wherein the frequency of the alternating electric field is between 100-500 kHz.
22. A method of preventing cancer cells in a subject from developing resistance to an alternating electric field, the method comprising:
prescribing a PTGER3 inhibitor to the subject; and
applying an alternating electric field to the cancer cell, wherein the frequency of the alternating electric field is between 100-500 kHz.
23. A method of restoring sensitivity to TTField in TTField-resistant cancer cells of a subject, the method comprising prescribing a PTGER3 inhibitor to the subject, wherein the sensitivity to TTField of the TTField-resistant cancer cells of the subject is restored after administering the PTGER3 inhibitor to the subject.
24. A method of reducing the viability of a TTField-resistant cancer cell in a subject, the method comprising:
recommending to the subject to administer an inhibitor of a target in the EP 3-controlled resistance pathway; and
applying an alternating electric field to the cancer cells of the subject, the frequency of the alternating electric field being between 100-500 kHz.
25. The method of claim 24 wherein the target in the EP 3-controlled resistance pathway is selected from ZNF488 and PRDM 8.
26. The method of claim 25, wherein the inhibitor is selected from azacytidine and decitabine.
Background
Tumor therapy field (TTField) is an effective anti-tumor therapy modality that is delivered by the non-invasive application of low intensity, medium frequency (e.g., 100-. TTField exerts directional forces on polar microtubules and interferes with the normal assembly of the mitotic spindle. Such interference with microtubule dynamics results in abnormal spindle formation and subsequent mitotic arrest or delay. Cells may die or progress to cell division at the time of mitotic arrest, leading to the formation of normal or abnormal aneuploid progeny. The formation of tetraploid cells may occur as a result of mitotic withdrawal through gliding, or may occur during inappropriate cell division. The abnormal daughter cells may die at a subsequent interval, may undergo permanent arrest, or may proliferate through additional mitosis, where they will undergo further TTField attack. The subject of Giladi et al,Sci Rep. 2015;5:18046。
in an in vivo environment, wearable and portable devices (optube) may be used®) Delivering TTField therapy. The delivery system includes an electric field generator, 4 adhesive patches (non-invasive, insulated transducer array), a rechargeable battery, and a carrying case. The transducer array is applied to the skin and connected to the device and battery. The treatment is designed to be worn for as many hours as possible throughout the day and night.
TTField may use, for example, Inovitro in a preclinical settingTMTTfield laboratory work table system bodyAnd (4) externally applying. InovitroTMIncluding a TTField generator and a substrate containing 8 ceramic disks per plate. Cells were plated on coverslips placed in each dish. TTField is applied using two pairs of orthogonal transducer arrays insulated by a high dielectric constant ceramic in each disk. The orientation of the TTField in each disc is switched by 90 ° every 1 second, thus covering a different axis of orientation for cell division.
Despite surgery and aggressive chemoradiotherapy, Glioblastoma (GBM) is the most common and most fatal malignant brain cancer in adults. The tumor treatment field (TTField) has been approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM. Addition of TTField resulted in a significant improvement in overall survival. TTField is a low intensity alternating electric field that is believed to disturb the assembly of mitotic macromolecules, leading to disrupted chromosome segregation and cell death. However, resistance to treatment developed in many TTField responders.
Several human GBM cell lines were developed which demonstrated relative resistance to the cytotoxic effects of TTField compared to the parental cells.
Disclosure of Invention
Methods of reducing the viability of TTField resistant cancer cells in a subject by recommending to the subject administration of a prostaglandin E receptor 3 (PTGER3) inhibitor and applying an alternating electric field to the cancer cells of the subject are provided. The frequency of the alternating electric field is between 100 and 500 kHz.
In some cases, methods are provided for preventing cancer cells of a subject from developing resistance to an alternating electric field by recommending to the subject to administer a PTGER3 inhibitor and applying the alternating electric field to the cancer cells of the subject. The frequency of the alternating electric field is between 100 and 500 kHz.
In some cases, methods are provided for restoring sensitivity to TTField in TTField-resistant cancer cells in a subject by recommending to the subject to administer a PTGER3 inhibitor. In this aspect, sensitivity to TTField is substantially restored in the TTField-resistant cancer cells of the subject.
In some cases, methods are provided for preventing a cancer cell of a subject from developing resistance to an alternating electric field by prescribing a PTGER3 inhibitor to the subject and applying the alternating electric field to the cancer cell. The frequency of the alternating electric field is between 100 and 500 kHz.
In some cases, a method of restoring sensitivity to TTField in TTField-resistant cancer cells in a subject by prescribing a PTGER3 inhibitor to the subject. In this aspect, the sensitivity of the TTField-resistant cancer cells of the subject to TTField is restored after the PTGER3 inhibitor is administered to the subject.
In some cases, methods are provided for reducing the viability of TTField-resistant cancer cells in a subject by recommending to the subject to administer an inhibitor of a target in the EP 3-controlled resistance pathway and applying an alternating electric field to the cancer cells of the subject. The frequency of the alternating electric field is between 100 and 500 kHz.
Drawings
Figure 1A shows an example of micronuclei development in a human LN827 Glioblastoma (GBM) cell line, comparing cells not treated with TTField to those treated with TTField (left panel) and shows the percentage of cells with micronucleus structure (histogram, right panel);
FIG. 1B provides an exemplary schematic showing how TTfield-produced micronuclei induce the STING pathway and cause cell apoptosis in abnormal mitosis, chromosome instability and production of proinflammatory cytokines and type I interferons;
figure 2 shows an exemplary procedure for developing TTField resistant glioblastoma cells with increased resistance to TTField as indicated by an increase in cell number;
FIG. 3 shows the results of an exemplary experiment demonstrating that U87 GBM resistant cells do not change in cell size and that the cells form micronuclei in response to TTfield, but no longer produce the STING pathway pro-inflammatory cytokine response;
FIG. 4 shows that TTField resistant GBM cells (U87R, LN428R, and LN827R) maintain the resistant phenotype upon depletion of STING and AIM;
fig. 5 and 6 show exemplary changes in overall network gene expression using NETZEN (a proprietary AI algorithm) in resistant GBM cells shortly after TTField exposure;
FIGS. 7A-7B show that PTGER3 is the highest-ranking master regulator of resistance in all three GBM TTField resistant cell lines;
fig. 8 shows an exemplary 2D view of the core PTGER3 subnetwork in TTField resistant cells 24 hours after TTField treatment;
fig. 9 shows exemplary 2D views of the core PTGER3 subnetwork in TTField resistant cells after 1 and 5 weeks;
FIG. 10 shows that PTGER3 upregulation correlates with TTfield-induced STING and activation of apoptosis in the initial exposure to TTfield in three GBM cell lines using quantitative PCR;
FIG. 11 shows that upregulation of PTGER3 in the rat GBM model correlates with TTfield-induced STING pro-inflammatory cytokines;
FIG. 12 shows an exemplary schematic of the PTGER3 (also known as EP3) signaling pathway;
figure 13 shows that PTGER3 inhibitor (aspirin) reduced resistance to TTField in TTField-resistant GBM cells;
FIG. 14 shows that PTGER3 inhibition restores the sensitivity of GBM resistant cells to TTField;
FIGS. 15A-15B show that forced PTGER3 expression in TTfield-sensitive GBM cells confers resistance to TTfield;
figure 16 shows that PTGER3 inhibition in TTField-sensitive GBM cells prevents development of resistance to TTField;
FIG. 17 shows that PTGER3 is up-regulated and translocated to or present in the nucleus (as shown by DAPI and limited by lamin A/C) upon initial exposure to TTField;
FIGS. 18A-18B show the stem cell-rich phenotype (stemness) of TTField-resistant GBM cells (evidenced by increased glioma sphere (gliomasphere) formation and CD44 surface markers); and
figure 19 shows that TTField resistant cells are enriched for GBM stem cells, resulting in increased tumor growth and death.
Detailed Description
TTField is an effective anti-tumor treatment modality that is delivered by the non-invasive application of low intensity, medium frequency (e.g., 100-. However, in certain cases, tumor cells may develop resistance to TTField treatment, resulting in reduced efficacy in these cases. In some aspects, resistance to TTField is referred to as an increase in "stem cell characteristics" or phenotype similar to stem cells. Stem cell characteristics can be measured by expression of a stem cell characteristic marker (e.g., CD44) and by the ability to form a brain tumor when grown in serum-free media in 3D spheres and implanted into the brain of an immunosuppressed mouse.
Importantly, TTField-induced chromosomal instability (e.g., formation of cytoplasmic micronuclei) was retained in resistant cells compared to their sensitive counterparts, indicating that resistance to TTField is mediated by abiotic physical mechanisms. Indeed, the inflammatory response induced by TTField is severely inhibited in resistant cells, supporting the hypothesis that resistance to TTField is conferred by the selective loss of the detrimental effects induced by biophysical injury. This acquired TTField resistance phenotype is associated with a shift to a stem cell-like state as determined by standard neurosphere assays.
Recently, the cyclic GMP-AMP synthase (cGAS) -interferon gene stimulator (STING, encoded by TMEM 173) pathway of the immunosensing molecule was identified as an important component of cytoplasmic DNA sensing and plays an important role in mediating the immune response in cells. Ghaffari et al, British Journal of Cancer, vol 119, pp 440-449 (2018); see, for example, fig. 3. Activation of the STING pathway mediates an immune response in response to abnormalities in the cell (e.g., the presence of cytoplasmic double stranded dna (dsdna)).
Prostaglandin E receptor 3 (PTGER3) is one of four receptors, the G-protein coupled receptor and prostaglandin E2. PTGER3 is implicated in biological systems and diseases associated with inflammation, cancer, digestion, nervous system, renal function, blood pressure and uterine contractions.
PTGER3 inhibitors include NSAIDs (e.g., aspirin, ibuprofen), cox2 inhibitors (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG 041. NSAIDs are common over-the-counter drugs used to relieve pain and reduce inflammation.
Aspects described herein use a systematic approach to understand the "stem cell trait" development in resistant cells and identify the primary regulators of the resistance mechanism with the aid of a suite of innovative computing platforms. Three networks were found to be disrupted, including nervous system developmental regulation, inflammatory response, and cell-cell adhesion, all of which play a role in GBM stem cell-like cells.
Using a unique master regulator hierarchy, prostaglandin E receptor 3 (PTGER3) was identified as a key master regulator at the vertices of these pathways and is responsible for the TTField resistance phenotype. PTGER3 is rapidly upregulated in GBM cells upon exposure to TTField and directs the treated cells away from the beneficial inflammatory pathways that TTField also activates in parallel.
The PTGER3 signaling pathway is upregulated by interacting with prostaglandin E2 (PGE 2). The combination of TTField and aspirin or other traditional NSAIDs (e.g., cox2 inhibitors) may prevent PGE2 biosynthesis and thus activation of PTGER3 signaling. Alternatively, PTGER3 receptor antagonists (e.g., L798,106,106, ONO-AE3-240, and DG-O41) may be used alone or in combination with other PTGER3 antagonists and inhibitors. Such combinations can be used to restore sensitivity to TTField in cells that develop resistance.
Furthermore, GBM cells treated with PTGER3 inhibitors may prevent the cells from developing resistance to TTField when exposed to TTField, e.g., after about 3 weeks to more than 5 weeks.
Methods of reducing the viability of TTField resistant cancer cells in a subject by recommending to the subject administration of a prostaglandin E receptor 3 (PTGER3) inhibitor and applying an alternating electric field to the cancer cells of the subject are provided. The frequency of the alternating electric field may be between 100 and 500 kHz.
The term "reducing viability" as used herein refers to reducing proliferation, inducing apoptosis, or killing cancer cells. The term "TTField resistant cancer cell" as used herein means that the cancer cell shows a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% decrease in sensitivity to TTField treatment compared to TTField sensitive cancer cells. The term "sensitivity" as used herein refers to responsiveness to TTField treatment, e.g., as measured by a decrease in cell number after treatment with TTField.
The term "recommendation" refers to a recommendation or indication from, for example, a medical professional (e.g., doctor, physician's assistant, nurse, caregiver, etc.) for a subject (e.g., patient).
In some cases, the PTGER3 inhibitor is selected from one or more of an NSAID (e.g., aspirin, ibuprofen), a cox2 inhibitor (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG 041. In one aspect, the PTGER3 inhibitor is aspirin.
In some cases, the recommended concentration of PTGER3 inhibitor in the subject is about 1-500 nanomolar for L798,106, 0.1-2 millimolar for aspirin, 0.5-50 nanomolar for DG041, or 1-500 nanomolar for celecoxib. The term "recommended concentration" may refer to a recommended dose sufficient to provide an intermittent or sustained level of PTGER3 inhibitor in a subject. In some cases, the recommended concentration of PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks. In some cases, the cancer cell is selected from glioblastoma, lung, pancreatic, mesothelioma, ovarian, and breast cancer cells.
A further aspect provides a method of preventing a cancer cell of a subject from developing resistance to an alternating electric field by recommending administration of a prostaglandin E receptor 3 inhibitor to the subject and applying the alternating electric field to the cancer cell of the subject. The frequency of the alternating electric field is between 100 and 500 kHz. In some cases, the frequency of the alternating electric field is between 100 and 300 kHz.
In some cases, the PTGER3 inhibitor is selected from one or more of an NSAID (e.g., aspirin, ibuprofen), a cox2 inhibitor (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG 041. In one aspect, the PTGER3 inhibitor is aspirin.
In some cases, the recommended concentration of PTGER3 inhibitor in the subject is about 1-500 nanomolar for L798,106, 0.1-2 millimolar for aspirin, 0.5-50 nanomolar for DG041, or 1-500 nanomolar for celecoxib. The term "recommended concentration" may refer to a recommended dose sufficient to provide an intermittent or sustained level of PTGER3 inhibitor in a subject. In some cases, the recommended concentration of PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks. In some cases, the cancer cell is selected from glioblastoma, lung, pancreatic, mesothelioma, ovarian, and breast cancer cells.
A further aspect provides a method of restoring sensitivity to TTField in TTField-resistant cancer cells in a subject by recommending to the subject administration of a PTGER3 inhibitor, wherein sensitivity to TTField in TTField-resistant cancer cells in the subject is substantially restored.
The term "restoring sensitivity" refers to reestablishing the responsiveness of TTField resistant cells as the responsiveness of TTField sensitive cells. In this regard, "responsiveness" is measured by counting the number of cells before and after exposure to TTField. The term "substantially recovering" refers to increasing the responsiveness of TTField resistant cells. In some cases, the responsiveness of TTField resistant cells is restored by at least 10%. In some cases, the responsiveness of TTField resistant cells is restored by at least 25%. In some cases, responsiveness of TTField resistant cells is restored by at least 50%.
In some cases, the PTGER3 inhibitor is selected from one or more of an NSAID (e.g., aspirin, ibuprofen), a cox2 inhibitor (e.g., celecoxib, valdecoxib, rofecoxib), L798,106, and DG 041. In one aspect, the PTGER3 inhibitor is aspirin.
In some cases, the recommended concentration of PTGER3 inhibitor in the subject is about 1-500 nanomolar for L798,106, 0.1-2 millimolar for aspirin, 0.5-50 nanomolar for DG041, or 1-500 nanomolar for celecoxib. The term "recommended concentration" may refer to a recommended dose sufficient to provide an intermittent or sustained level of PTGER3 inhibitor in a subject. In some cases, the recommended concentration of PTGER3 inhibitor in the subject is maintained for at least about 3 days to 5 weeks. In some cases, the cancer cell is a glioblastoma cell.
Yet another aspect provides a method of reducing the viability of TTField-resistant cancer cells in a subject by prescribing a PTGER3 inhibitor to the subject and applying an alternating electric field to the cancer cells. The frequency of the alternating electric field may be between 100 and 500 kHz.
The term "prescribing" as used herein refers to a medical professional authorized to write a prescription, either providing a prescription of medication to a subject or communicating the prescription to a pharmacy or other pharmaceutical pharmacy.
A further aspect provides a method of preventing a cancer cell of a subject from developing resistance to an alternating electric field by prescribing a PTGER3 inhibitor to the subject and applying the alternating electric field to the cancer cell. The frequency of the alternating electric field may be between 100 and 500 kHz.
Yet another aspect provides a method of restoring sensitivity to TTField in TTField-resistant cancer cells of a subject by prescribing a PTGER3 inhibitor to the subject, wherein the sensitivity to TTField of the TTField-resistant cancer cells of the subject is restored after administering the PTGER3 inhibitor to the subject.
In some cases, methods are provided for reducing the viability of TTField-resistant cancer cells in a subject by recommending to the subject to administer an inhibitor of a target in the EP 3-controlled resistance pathway and applying an alternating electric field to the cancer cells of the subject. The frequency of the alternating electric field is between 100 and 500 kHz.
In some cases, the target in the resistance pathway controlled by EP3 is selected from ZNF488 and PRDM 8. Examples of PRDM8 inhibitors include, but are not limited to, azacytidine and decitabine.
As shown in figure 1A, human LN827 glioblastoma cells were treated with TTField at 200 kHz for 24 hours, then fixed with 4% PFA for 20 minutes. Cells were stained with DAPI (4', 6-diamidino-2-phenylindole) at a dilution of 1:5000 and incubated for 5 minutes at room temperature to stain nuclei and micronuclei. Micronuclei (arrows) can be seen in TTField treated cells (lower panel). The histogram shows an increase in micronucleus structure of about 15%.
FIG. 1B depicts the induction of pro-inflammatory STING and cellular apoptosis pathways by dsDNA (double-stranded DNA). dsDNA can be produced from micronuclei induced by abnormal mitosis. Abnormal mitosis can be induced, for example, by TTField. TTField can also reduce nuclear envelope integrity as shown by the disruption of lamin B1 structure leading to induction of dsDNA and STING pathways in the cytoplasm, as shown.
For the experiments summarized in FIG. 2, a TTfield resistant human GBM cell line was generated by seeding LN827 cells at a density of 10,000/ml and treatment with TTfield at a frequency of 200 kHz for repeated 1-week cycles. For each cycle, cells were counted on days 2, 4 and 7. At the end of each cycle, cells were again seeded at the same density as day 0, harvested and processed for RNAseq and cryopreserved for future analysis. Resistant cells appeared after at least four weeks of TTField treatment. As shown in fig. 2, the cell number of TTField treated cells was significantly higher after week 5 compared to the previous weeks, demonstrating the development of cell resistance to the cell number reducing effect of TTField in non-resistant cells.
Human GBM cell lines were treated with and without TTField for 1 week at a frequency of 200 kHz (TTF = sensitive cells; R-TTF = resistant cells) (fig. 3). Cells were analyzed for size (flow cytometry), micronucleus structure (immunofluorescence), and type 1 interferon response gene (qPCR). As shown in the left panel, U87C (sensitive) and U87R (resistant) showed no change in cell size. However, the right panel (top) shows that the micronuclei is still formed (compare TTF and R-TTF). Interferon stimulated gene 15 (ISG15), a key type 1 interferon response gene, is no longer produced in U87R and LN827R resistant cell lines.
Figure 4 shows that the pro-inflammatory and resistance pathways induced by TTField are independent. Using shRNA (short hairpin RNA), the STING/AIM2 pathway was depleted in "knock-out" (KD) TTField resistant cell lines (U87R, LN428R, LN 827R). Cells were treated for 3 days as indicated and cell number was determined by a cell counter (Bio-Rad TC 10). As shown in fig. 4, resistance to TTField was maintained even when the STING/AIM2 pathway was inhibited by shRNA (e.g., TTField column compared to double KD + TTField).
Figure 5 shows exemplary changes in overall gene expression changes in control cells and in resistant cells (LN428, LN827 and U87) at 1 and 5 weeks after TTField exposure.
FIG. 6 shows the overall gene network changes in resistant GBM cells using algorithms and the correlation of identified genes with stem cell trait-associated phenotypes (e.g., ERG, FOXF1, NFKBIZ, LIF, BCL3, EHF, ZNF488, SLC2A4RG, ETV4, PTGER3) and immune responses (e.g., CEBPD, RCOR2, TRIM22, SLC1A3, PLSCR1, FLI 1).
Fig. 7A shows nSCORE ratings of the top major regulator of resistance to TTField identified using NETZEN (fig. 7A) across 4 different time points (0 hr, 6 hr, 24 hr, 1 week, 5 weeks) in 3 different GBM cell lines. Figure 7B shows that RNAseq and western blots determined for PTGER3 expression correlate with PTGER3 nSCORE ranking. EP3 overexpression in the 293T cell line was used as a positive control, and β -actin as a loading control.
Fig. 8 provides a two-dimensional view of the base factor network controlled by PTGER3 after 24 hours of exposure to TTField. Figure 9 shows how the gene network controlled by PTGER3 becomes dominant as resistant cells take over the cell culture (week 1+ week 5).
Figure 10 shows that PTGER3 upregulation in 3 is associated with TTField-induced STING and activation of cellular apoptosis following TTField exposure. The transcript levels of PTGER3, IL6 and ISG15 (markers of STING and cell apoptosis activation) were tested using quantitative RT-PCR.
Figure 11 shows that PTGER3 upregulation in vivo in the rat GBM model correlates with ttifield-induced STING proinflammatory cytokines. Established by Novocure. TTField treatment was initiated in the F98 rat orthotopic GBM model (Novocure) 1 week after injection and continued for 1 week. The animals were then euthanized and tumor RNA was collected. Quantitative RT-PCR was performed to detect the transcript levels of PTGER3, IL6 and ISG 15.
FIG. 12 illustrates the PTGER3 pathway, including the portion of the signaling pathway affected by aspirin and the PTGER3 inhibitors L798,106 and DG041), based on the description from Markovic et al, Structural features of subtype-selective EP receiver modules, Drug Discovery Today, Vol.22 (1) to 1/1 of 2017.
Aspirin significantly reduced resistance to TTField in TTField resistant cells. Fig. 13 shows that aspirin reduced resistance to TTField in TTField-resistant GBM cells (U87R, L428R, and LN827R) (compare vehicle + TTField to aspirin + TTField). For the experiments summarized in fig. 13, resistant human GBM cells were treated with vehicle control or aspirin and with or without TTField for 3 days. The drugs were replenished daily. The number of viable cells was quantified at the end point using a cell counter.
Thus, patients who develop resistance to TTField treatment (e.g., during prolonged use) may be given aspirin (e.g., daily) to reduce resistance to TTField, making TTField treatment more effective over a longer period of time. Without being bound by theory, it is believed that this approach may improve the effectiveness of TTField in patients developing resistance.
For example, in U87R resistant cells, TTField decreased the viable cell count from 150K/disc to 125K/disc. When aspirin was provided to the cells, the viable cell count decreased from 150K/disc to 100K/disc. In LN428R resistant cells, TTField decreased the viable cell count from 85K/disc to 70K/disc. When the cells were provided with aspirin, the viable cell count decreased from 85K/disc to 40K/disc. In LN827R cells, TTField slightly increased the viable cell count. When the cells were provided with aspirin, the viable cell count decreased from approximately 125K/disc to 90K/disc.
PTGER3 inhibitors can restore sensitivity to TTField. Resistant human GBM cells were treated with vehicle control or EP3 (PTGER3) inhibitor L798,106 (left panel) or DG041 (right panel) with or without TTField at 200 kHz, respectively, for 3 days (fig. 14). DG041 was obtained from US Biological and L789,106 was obtained from Tocris. For the experiment summarized in fig. 14, the drugs were replenished daily. The number of viable cells was quantified at the end point using a cell counter. As shown in figure 14, PTGER3 inhibitors restored sensitivity to TTField (compare TTField columns to L798,106+ TTField and DG041+ TTField).
As shown in figure 14 (left panel), TTField reduced the viable cell count from about 200K/disc to 125K/disc for U87 GBM resistant cells. When the PTGER3 inhibitor L798,106 (0.5 μ M) was provided to the cells, the viable cell count decreased from about 200K/disc to 25K/disc. As shown in fig. 14 (right panel), TTField reduced the viable cell count from about 160K/disc to 100K/disc for U87 GBM resistant cells. When the PTGER3 inhibitor DG041 (50 nM) was provided to the cells, the viable cell count decreased from about 160K/disc to about 40K/disc. This data demonstrates that PTGER3 inhibitors can restore sensitivity to TTField even after resistance to TTField has developed.
Forced PTGER3 expression in TTField-sensitive GBM cells confers resistance to TTField. As shown in fig. 15A, human GBM cells were transduced with lentiviruses expressing either empty vector control (EV) or PTGER3 and subsequently treated with TTField at 200 kHz for 3 days. The number of viable cells was quantified using a cytometer. The EP3 overexpression efficiency was determined by western blotting (fig. 15B). Resistance was conferred only in cells overexpressing EP3 (U87 and LN428), but not in cells overexpressing EP3 that failed (LN 827).
The resistant cell line generation experiment using the original human GBM cell line from figure 2 was repeated with two additional groups including PTGER3 inhibitor (L798,106 and L798,106+ TTField) (figure 16). Inhibitors of L798,106 PTGER3 prevent the development of resistance to TTField. Cells were inoculated at the same cell density and time point and counted. Each cycle lasted 7 days for a total of 5 cycles.
The results shown in fig. 17 demonstrate the presence of EP3 in the core upon exposure to TTField. These results show that EP3 (a 7 transmembrane cell surface receptor) is present or translocated to the nucleus where it acts as the primary regulator of resistance to TTField, interacting directly with hundreds of genes. The vast majority of the major regulators are nuclear-localized transcription factors.
Without being bound by theory, it is believed that EP3 is upregulated and is present or translocated to the nucleus upon exposure to TTField, providing a mechanism whereby EP3 can modulate other genes directly or indirectly through other transcription factors (e.g., neuronal stem factor ZNF 488). Thus, EP3 is believed to modulate resistance to TTField by promoting the development and enrichment of GBM stem cells, which are resistant to many forms of therapy (e.g., TTField) due to their slow rate of recirculation and other survival and anti-apoptotic pathways.
As shown in fig. 18A-18B, TTField resistant GBM cells are enriched for a "stem cell characteristic" phenotype (e.g., glioma spherogenesis and CD44 surface markers). Figure 18A shows glioma sphere formation in resistant cells treated with TTField for 1 week, whereas TTField sensitive cells treated with TTField for 1 week did not show glioma sphere formation.
Figure 18B shows an increase in CD44 surface markers in TTField resistant cells compared to TTField sensitive cells. Human GBM cell lines were treated as indicated, then seeded at a density of 100 cells/well into 96-well plates in FBS-free stem cell culture medium, cultured for 4 weeks, and stained with calcein AM dye for 30 minutes at room temperature. Images of each well were taken in a plate reader (SpectraMax @ 3x) at a wavelength ex/em =456/541 nm. CD44 expression was measured by FACS.
As shown in fig. 19, TTField resistant cells were enriched for GBM stem cells, resulting in increased tumor growth and death. TTField resistant and TTField sensitive cells were implanted into mouse brains. An equal number of cells under each treatment condition were implanted in situ into the brain of NSG mice and survival was measured as a proxy for tumor growth.
Although the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the invention not be limited to the described embodiments, but that it have the full scope defined by the language of the following claims, and equivalents thereof.