Oncolytic herpes simplex virus infected cells
11612626 · 2023-03-28
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
A61M37/00
HUMAN NECESSITIES
A61K9/0009
HUMAN NECESSITIES
A61K41/00
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
A61K35/15
HUMAN NECESSITIES
A61K47/6901
HUMAN NECESSITIES
C12N5/0645
CHEMISTRY; METALLURGY
A61K9/5068
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A01N63/00
HUMAN NECESSITIES
A61K35/15
HUMAN NECESSITIES
A61K41/00
HUMAN NECESSITIES
A61K47/69
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
A61K9/50
HUMAN NECESSITIES
A61M37/00
HUMAN NECESSITIES
Abstract
A monocyte, monocyte derived cell or macrophage infected with an oncolytic herpes simplex virus is disclosed together with uses of such infected cells in the treatment of diseases such as cancer.
Claims
1. An isolated ex vivo cell productively infected with an oncolytic herpes simplex virus wherein the cell is a macrophage and wherein the oncolytic herpes simplex virus is HSV1716 or a mutant thereof, wherein the oncolytic herpes simplex virus mutant is an ICP34.5 null mutant.
2. The cell of claim 1, further comprising super-paramagnetic iron oxide nanoparticles.
3. A method of treating a disease in a subject in need of treatment, the method comprising administering to said subject a preparation comprising a population of oncolytic herpes simplex virus-infected cells of claim 1.
4. The method of claim 3, wherein the disease is cancer.
5. The method of claim 3, wherein the population of oncolytic herpes simplex virus-infected cells further comprises super paramagnetic iron oxide nanoparticles.
6. The method of claim 5, wherein the method further comprises applying a magnetic field to the subject in order to direct the population of cells of the administered preparation to a desired location in the subject's body.
7. The method of claim 6, wherein the desired location is the site of a tumor.
8. The method of claim 7, wherein the tumor is in an organ selected from the group consisting of the adrenal gland, adrenal medulla, anus, appendix, bladder, bone, bone marrow, brain, breast, cecum, central nervous system, brain, cerebellum, cervix, colon, duodenum, endometrium, gallbladder, esophagus, heart, ileum, intestines, jejunum, kidney(s), lacrimal gland, larynx, liver, lung(s), lymph, lymph node, mediastinum, mesentery, myometrium, nasopharynx, omentum, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, rectum, salivary gland, colon, small intestine, spleen, stomach, testis, thymus, thyroid gland, and uterus.
9. The method of claim 3, wherein the population of oncolytic herpes simplex virus-infected cells is prepared by contacting a population of cells comprising macrophages with a quantity of oncolytic herpes simplex virus under suitable conditions and for sufficient time to permit productive infection of the macrophages.
10. The method of claim 9, wherein the cells are maintained in culture under conditions in which the virus is able to induce cell death.
11. The method of claim 10, wherein the population of oncolytic herpes simplex virus-infected cells comprises a mixture of intact and lysed macrophages.
12. The method of claim 10, wherein 1-50% of the population of oncolytic herpes simplex virus-infected cells of the preparation are dying or dead.
13. A population of oncolytic herpes simplex virus-infected cells of claim 1, wherein the population comprises a mixture of intact and lysed macrophages.
14. A population of oncolytic herpes simplex virus-infected cells of claim 13, wherein 1-50% of the population of oncolytic herpes simplex virus-infected cells of the preparation are dying or dead.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
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(32) The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.
EXAMPLES
(33) The examples presented below show that tumour-conditioned macrophages infected with oncolytic HSV1716 (Seprehvir) display a classic activated (M1) profile characterized by the expression of pro-inflammatory factors such as iNOS, IL-6, IL-8 and TNF-α. Furthermore, the M1 macrophages can be magnetically labeled using super-paramagnetic iron oxide nanoparticles (SPIOs) and then non-invasively steered from the bloodstream into deep target tissues, including primary and secondary tumours, using pulsed magnetic-field gradients inherent to all magnetic resonance imaging systems (MRI). We have used this magnetic resonance targeting (MRT) approach to deliver a cell-based oncolytic virotherapy. Relaxometry measurements suggest that standard MR imaging can then be used to monitor the efficacy of this therapy.
Example 1
(34) HSV1716 and Human Primary Macrophages
(35) 1) In an initial study human macrophages were infected with HSV1716 at approximately 4 pfu/cell and the cells were then incubated under normal and hypoxic conditions. Samples were removed at various time points after infection (+1.5 hr, +24 hrs, +48 hrs and +72 hrs) and titrated (
(36) Within 1 hour 90% of the virus had been adsorbed by the macrophages and then no virus was detectable at 24 or 48 hrs in either normoxia or hypoxia (detection limit of titration is 100 pfu/ml).
(37) Significantly, virus was detectable at 72 hrs but the amounts at this time were similar in the normoxic vs hypoxic macrophages. This emergent virus is of significant interest as it could either be the original input which had entered some transient latent state or represent the first wave of replication in the macrophages.
(38) 2) Macrophages were infected with decreasing HSV1716 moi (40, 4, 0.4 and 0.04) and samples were titrated after 72 hrs only. Virus was detected from the macrophages infected at moi 40, 4 and 0.4 but not from those infected at 0.04 moi (
(39) In summary, human primary macrophages were found to have a high capacity to adsorb HSV1716, and active virus can be recovered from the macrophages after 48 hrs in culture.
Example 2
(40) Three different cancer cell lines were used (LNCaP, PC3, T47D) and macrophages were derived from human mononuclear cells; multicellular spheroids were prepared on agarose-coated culture plates; HSV1716GFP was used, to allow quantification of uptake by cancer cells and tumor spheroids by fluorescence microscopy and flow cytometry; finally, RT-PCR was performed to analyse changes in macrophage gene expression after HSV1716 infection.
(41) Results showed that HSV1716 induces cell death in prostate and breast cancer cell lines and that macrophages infected by HSV1716 are effectively killed within 96 hours; moreover, infiltration of spheroids with HSV1716-infected macrophages causes tumor spheroid cell death.
(42) Materials and Methods
(43) Cell Lines
(44) Human prostate carcinoma cell lines LNCaP and PC3 and human breast carcinoma cell line T47D were provided by Dr Helen Bryant (Department of Oncology, The Medical School, Sheffield, UK). Cells were cultured in RPMI supplemented with 10% Fetal Bovine Serum.
(45) Preparation of Human MDM
(46) Macrophages were derived from human mononuclear cells, which were isolated from platelet-depleted buffy coats (Blood Transfusion Service, Royal Hallamshire Hospital, Sheffield, UK). Mononuclear cells were separated from blood by using Ficoll gradient centrifugation (BURKE 2003). After isolation, mononuclear cells were seeded into T75 tissue culture flasks (˜70×10.sup.6 cells/flask) and cultured in IMDM supplemented with 2% AB serum for 3 days.
(47) Herpes Simplex Virus 1716
(48) HSV1716 was provided by Virttu Biologics (Glasgow, UK). Infection of tumor cells was performed by using multiplicity of infection (MOI), that is, the number of virus particles added per cell during infection, of 0.5 and 5. For macrophage infection, MOI of 5 and 50 were used. The labelling with GFP allowed detection of HSV1716 (measured by flow cytometry and fluorescent microscopy).
(49) Infection of Primary MDM
(50) MDM were cultured for 3 days in IMDM supplemented with 2% AB serum; after 3 days, cells were washed with PBS and medium was replaced with RPMI supplemented with 10% FBS. Cells were infected at MOI 50 and incubated overnight (normoxic conditions: 20% pO.sub.2; hypoxic conditions: 0.1% pO.sub.2). After 24 hours, conditioned medium was replaced with fresh medium and cells were incubated for further 72 hours. After 96 hours (4 days) from infection, cell viability was measured by flow cytometry.
(51) Infiltration of Primary MDM into Tumor Spheroids
(52) Tumor spheroids were prepared using LNCaP cells by seeding 2×10.sup.4 cells/well into 2% agarose-coated 96-well plates in 100 μl RPMI (+10% FBS). After 72 hours (3 days), 5×10.sup.3 infected macrophages were added to each well. Analysis of cell death was performed after a further 5 days; each day, spheroids were observed under the fluorescence microscope to detect the presence of HSV1716GFP in the hypoxic core.
(53) Flow Cytometry
(54) Cell viability/death and GFP expression were measured by flow cytometry. Cells were harvested, re-suspended in PBS and labelled with PI (1 μl/sample) to quantify cell death. Attune Acoustic Focusing Cytometer (Life Technologies) was used to analyse percentage of PI positive cells and GFP positive cells in each sample. PI (excitation wavelength: 488 nm; maximum emission: 617 nm) was detected by BL3 detector; BL1 was used for GFP detection (excitation at 488 nm and maximum emission at 509 nm).
(55) RT-PCR
(56) Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect RNA levels in infected macrophages and determine whether HSV1716 causes changes in gene expression. Cells (1.5×10.sup.6) were plated into 6-well plates and infected with HSV1716 at MOI 50. After incubation for 48 hours (normoxic conditions: 20% pO.sub.2; hypoxic conditions: 0.1% pO.sub.2), cells were harvested and RNA extraction was performed using the RNeasy Mini Kit (Qiagen). cDNA was synthesised from RNA using the Primer design Precision nanoScript RT Kit and the T100 Thermal Cycler (Bio-Rad). cDNA was plated in 384-well PCR plates with primers of genes of interest. PCR was performed using the ABI7900 Real Time PCR.
(57) TABLE-US-00001 IL-6 forward: (SEQ ID NO: 1) 5′-CGAAAGTCAACTCCATCTGCC-3′ reverse: (SEQ ID NO: 2) 5′-GGCAACTGGCTGGAAGTCTCT-3′ IL-8 forward: (SEQ ID NO: 3) 5′-GGGCCATCAGTTGCAAATC-3′ reverse: (SEQ ID NO: 4) 5′-TTCCTTCCGGTGGTTTCTTC-3′ TNFα forward: (SEQ ID NO: 5) 5′-CCAGGAGAAAGTCAGCCTCCT-3′ reverse: (SEQ ID NO: 6) 5′-TCATACCAGGGCTTGAGCTCA-3′) IL-1 forward: (SEQ ID NO: 7) 5′-CACCTCTCAAGCAGAGCACAG-3′ reverse: (SEQ ID NO: 8) 5′-GGGTTCCATGGTGAAGTCAAC-3′) NFκB forward: (SEQ ID NO: 9) 5′-ACCTGAGTCTTCTGGACCGCTG-3′ reverse: (SEQ ID NO: 10) 5′-CCAGCCTTCTCCCAAGAGTCGT-3′ TGFβ forward: (SEQ ID NO: 11) 5′-TAGGAACAGGCGGCGACGAATACA-3′ reverse: (SEQ ID NO: 12) 5′-CACAATCACAAGGCAACTTCAAT-3′ IL-10 forward: (SEQ ID NO: 13) 5′-GCCTAACATGCTTCGAGATC-3′ reverse: (SEQ ID NO: 14) 5′-CTCATGGCTTTGTAGATGCC-3′ VEGF-A forward: (SEQ ID NO: 15) 5′-GAAGTTCATGGACGTCTACCAG reverse: (SEQ ID NO: 16) 5′-CATCTGCTATGCTGCAGGAAGCT-3′ CXCL-6 forward: (SEQ ID NO: 17) 5′-GAATTTCCCCAGCATCCCAAAG-3′ reverse: (SEQ ID NO: 18) 5′-TGCCTTCTGCACTCCCTTTATC-3′ CXCL-1 forward: (SEQ ID NO: 19) 5′-AGAATGTTTTCAAATGTTCTCCAGTC-3′ reverse: (SEQ ID NO: 20) 5′-GGCCATTTGCTTGGATCCG-3′
Statistical Analysis
(58) Data are reported as mean±SEM. Statistical analysis and graphics were performed using GraphPad Prism. Two-way ANOVA test for multiple comparisons and multiple t tests were performed to compare experimental data obtained. Statistical significance was limited to the value of p=0.05.
(59) Results
(60) HSV1716 Induces Tumor Cell Death
(61) LNCaP Cell Line
(62) To analyse the oncolytic potentiality of HSV1716 on prostate cancer cells, LNCaP cells were seeded into 12-well plates (2×10.sup.4 cells/well) and infected with HSV1716 at MOI 0 (control), 0.5 and 5. The infection was repeated using HSV1716-GFP in order to visualise virus uptake by living cells. Plates were kept in normoxic and hypoxic incubators, to investigate the ability of HSV1716 to kill cells in both oxygenated and non-oxygenated conditions. After 24 hours, conditioned medium was replaced with fresh medium. After 72 hours, plates were harvested and cells were analysed by flow cytometry. Cell death was measured as PI(+) cells; virus uptake in living cells was measured as GFP(+)/PI(−) cells (
(63) PC3 Cell Line
(64) PC3s are a prostate cancer cell line with high metastatic capacity and considerably more aggressive than LNCaPs. 2×10.sup.4 cells were seeded into 12-well plates and infected with HSV1716 at MOI 0, 0.5 and 5. Cells were incubated in normoxic and hypoxic conditions for 72 hours. After 24 hours, conditioned medium was replaced with fresh medium to eliminate viral particles not taken up by cells. After 72 hours, cells were analysed by flow cytometry and the amount of PI(−)/GFP(+) cells was plotted (
(65) T47D Cell Line
(66) To investigate the oncolytic ability of HSV1716 on different types of solid tumors, effects of infection of a breast carcinoma cell line, T47D, was evaluated. 1×10.sup.5 cells were seeded into 12-well plates and infected with HSV1716 and HSV1716-GFP at MOI 0, 0.5 and 5. Cells were incubated under normoxic and hypoxic conditions for 72 hours and analysed by flow cytometry. No signs of cell death were observed in any conditions, while virus uptake was markedly high even at MOI 0.5 (data not shown). Therefore, the analysis was repeated after 120 hours, to verify whether T47D cell line is either not responsive to the HSV1716, or just less sensitive than prostate carcinoma cell lines. After 120 hours, cells were observed by flow cytometry and the amount of PI(−)/GFP(+) cells was plotted (
(67) Effects of HSV1716 Infection on Human Macrophages
(68) HSV1716 Effectively Kills Human Macrophages
(69) To determine if macrophages could be used as a delivery system for HSV1716 therapy, it was fundamental to test the consequences of viral infection on macrophage cells. After 3 days from isolation, MDM were harvested and counted; 1×10.sup.6 cells were seeded in 6-well plates. Once cells attached to the plastic, infection was performed. HSV1716 was added at MOI 0 (control), 5 and 50. Cells were incubated in normoxic and hypoxic conditions for 96 hours. Medium was replaced with fresh medium after 24 hours, and on day 4 supernatant was collected from each well for further studies. After 96 hours, plates were harvested and cell viability was analysed by flow cytometry. Results showed an effective killing of cells at both MOI 5 and 50, with high percentages of cell death under both normoxic conditions (63±2.76% at MOI 5, 62±2.42% at MOI 50, p<0.0001) and hypoxic conditions (43±4.91% at MOI 5, p<0.001, and 57±7.34% at MOI 50, p<0.0001) (
(70) Infected Macrophages Release Viral Particles
(71) Since more than half of cells are killed by HSV1716, the considerable viral replication following infection and lysis of cells should lead to the release of viral particles in the microenvironment. Therefore, to confirm the ability of HSV1716 to kill and replicate in macrophages, supernatant from each well was collected after 96 hours from infection and analysed. Samples, consisting of medium acquired from cells infected at M010, 5 and 50 in both normoxic and hypoxic conditions and the presence of viral particles in the supernatant was determined by titration. HSV1716 was detected in the supernatant of MDM infected at MOI 5 and 50, with higher concentration for the higher MOI, while no virus was observed in control groups (Table 1 (
(72) To reaffirm this result, infection of tumor cells with conditioned medium collected from infected MDM was performed. LNCaP cells were seeded into 12-well plates (1×10.sup.4 cells/well) and infected with 100 μl of MDM supernatant in 1 ml RPMI. After 120 hours from infection, cells were harvested and analysed by flow cytometry. After PI staining, however, cell death was found to be only significant at MOI 50, in cells infected with conditioned medium collected from MDM under normoxic conditions (40±7.26%, p<0.01), in contrast with what was expected from titration studies (higher cell death under hypoxic conditions) (
(73) HSV1716 Infection Modifies Macrophage Gene Expression
(74) To investigate how infection with HSV1716 changes MDM gene expression, mRNA levels of cytokines and growth factors of interests were quantified using quantitative RT-PCR. 1.5×10.sup.6 cells were seeded into 6-well plates and infected at MOI 50; plates were incubated under normoxic and hypoxic conditions, to understand possible alterations in gene expression due to the different environment. 48 hours post infection, cells were harvested and mRNA was extracted from infected cells and control groups. cDNA was then synthesised from RNA, and plated into 384-well plates with primers of genes of interest: the pro-inflammatory cytokines IL-6, IL-8, TNF-α, IL-1, CXCL1; the transcription factor NFκB, the anti-inflammatory cytokines IL-10 and CXCL-6, the growth factors VEGF-A and TGF-β. β-actin was chosen as constitutively expressed housekeeping gene. After performing RT-PCR, mRNA levels of each gene were normalised for β-actin concentration and fold changes in the expression were calculated. Gene induction profile caused by HSV1716 infection was obtained in duplicate. Fold changes in expression calculated for each gene were reported; results suggest that, at MOI 50, HSV1716 is an inducer of pro-inflammatory cytokines (higher induction profile of IL-8, IL-1 and the pro-inflammatory transcription factor NFκB in hypoxic conditions). At the same time, expression of NFκB and the anti-inflammatory TGF-β and IL-10 were reduced in normoxia (despite an apparent induction of the latter under hypoxic conditions), while no detectable alteration of gene expression was observed for the chemokines CXCL-1 and CXCL-6. Interestingly, a markedly high induction of VEGF-A after infection was observed under hypoxic conditions (Table 2 (
(75) Infiltration of Spheroids with MDM Leads to Tumor Shrinkage
(76) To investigate if delivery of HSV1716 to tumors and, specifically, to the hypoxic core can be mediated by the use of macrophages, tumor spheroids were generated. The use of spheroids has the advantage, compared to 2D cultures, of being constituted by an oxygen-depleted central area surrounded by a well-oxygenated zone; therefore, a spheroid mimics 3D tumors. Tumor spheroids were generated on day 1 using LNCaP cells (1.5×10.sup.4 cells seeded into 2% agarose-coated 96-well plates). 3 days after plating cells (day 4), spheroids of 800 μm/1 mm diameter had developed. MDM were infected with HSV1716 at MOI 50 on day 3 and incubated for 24 hours, non-infected cells were used as controls. On day 4, cells were harvested and counted; spheroids were infiltrated with 5×10.sup.3 MDM, both at MOI 0 (control MDM) and MOI 50 (infected MDM). In addition, control spheroids (non-infiltrated) were taken into account. Plates were incubated for further 120 hours (until day 9). On day 6, after 72 hours from MDM infection, pictures were taken using a fluorescence microscope, to visualise the presence of HSV1716 (labelled with GFP) inside the spheroids. Images revealed the presence of HSV1716-infected MDM; however, MDM seemed to be confined to the viable rim which surrounds the hypoxic core, while no GFP(+) cells were observed in the inner areas of spheroids. Central areas of spheroids (control groups) were found to be slightly necrotic and markedly darker than what was expected. On day 9, spheroids were further observed under the microscope and pictures were taken; no significant alterations in the shape and size of spheroids were detected. Spheroids were harvested and washed after 5 days from infiltration (day 9) and cell viability was analysed by flow cytometry; while no significant cell death was observed in control groups and in spheroids infiltrated with non-infected MDM, infiltration with MDM infected at MOI 50 caused oncolysis of 51±5.92% of cells (p-value<0.01) (
(77) Discussion
(78) The findings revealed that prostate cancer cell lines LNCaP and PC3 and breast cancer cell line T47D are sensitive to HSV1716, indicating that this virus could be used as a therapy to treat a broad range of tumors. Although at different extent, both prostate and breast cancer cell lines were responsive to HSV1716 infection at MOI 5 after 72 hours (LNCaP, PC3) and 120 hours (T47D); in addition, the detection of high levels of virus uptake in living cells suggests that further cytotoxic effect could be induced over time. Percentages of cell death were similar in normoxic and hypoxic conditions for all the cell lines tested (no statistical significance was observed between the two groups): this result indicates that hypoxia does not confer resistance to HSV1716 to tumor cells in vitro; HSV1716, therefore, could potentially be used to kill difficult to treat hypoxic areas of cancer. Interestingly, despite the fact that significant cell death is observed under oxygen-depleted conditions, virus uptake in hypoxia is generally lower than the uptake in normoxia for both prostate and breast cancer cell lines—and is not significant for PC3. However, the high levels of cell death reported suggest a greater sensitivity to HSV1716 in hypoxic conditions.
(79) The studies carried out on human macrophages showed that they are sensitive to HSV1716. The ability of HSV1716 to kill macrophages after 96 hours from infection has a fundamental importance, as it implies the possibility of exploiting MDM as a delivery system for the virus, which will be transported inside the hypoxic areas of tumors by MDM, replicate, lyse MDM and, subsequently, infect and kill the nearby cancer cells. To further demonstrate that HSV1716 replication in MDM leads to the release of viral particles in the microenvironment, supernatant was collected from infected macrophages, with the aim of analysing it and detecting the presence of HSV1716. Results revealed that the viral concentration increases with an increasing MOI, as expected; interestingly, under hypoxic conditions replication and release of viral particles were 3 fold greater than in normoxia. When LNCaPs were infected with the same supernatant, however, after 120 hours cell death was only significant in normoxia, at M0150, whereas no significant values were observed under hypoxic conditions or at lower MOI. This result could be explained by the fact that the amount of viral particles observed after titration of supernatant, although higher in hypoxia, is generally not greater than 3×10.sup.3 PFU/ml: such quantity could be not sufficient for the virus to kill cells, considering that 100 μl of supernatant containing HSV1716 at 3×10.sup.3 PFU/ml (or less, in case of MOI 5) were used to infect 2×10.sup.4 cells; this means that LNCaPs were infected at MOI<0.05—an extremely low MOI (bearing in mind that, when performing infection with HSV1716, significant cell death was only observed at MOI 5). However, such low values of HSV1716 detected in MDM-conditioned media implies release of the virus, and the importance of this finding is clear when considering that, in a putative therapeutic approach, once delivered through MDM and released in the environment, viral particles would encounter tumor cells, infect them, replicate, further amplifying the amount of viral copies, and subsequently disseminate widely into tumors.
(80) To test the ability of MDM to deliver HSV1716 to tumors, and specifically to hypoxic areas, multicellular 3D spheroids were prepared, with the aim of mimicking the structure of a real tumor. The main objective was to understand if MDM actually deliver the virus, sufficiently for cell death to be induced in 3D spheroids. The relatively large diameter of spheroids (800 μm−1 mm) allowed the observation of possible alterations in shape and size under the microscope. In addition, the presence of GFP-labelled HSV1716 gave the opportunity to detect the presence of infected MDM inside the spheroids and, therefore, to observe if MDM actually reached the hypoxic core.
(81) Significant cell death was observed in spheroids infiltrated with infected MDM (MOI 50) compared to control groups (p-value=0.009) and spheroids infiltrated with non-infected MDM (p-value=0.004).
(82) HSV1716GFP was observed in spheroids treated with infected MDM, after 72 hours from MDM infection (day 6) with weak gfp fluorescence co-localising with the oxygenated rim. The absence of pronounced green stains could be due to the small quantity of MDM used (only 5×10.sup.3 cells were infiltrated with each spheroid). However, delivery of HSV1716 was successful as spheroids infiltrated with infected MDM showed significant levels of cell death (51±5.92%, p-value<0.01) suggesting that HSV1716 replicates inside MDM and spreads in the micro-environment, ultimately killing tumor cells.
(83) To understand how HSV1716 infection modifies gene expression in MDM, RT-PCR was performed. Genes of interest were selected based on their immune properties, the pro-inflammatory cytokines IL-8, IL-6, TNF-α, CXCL-1, the anti-inflammatory cytokines IL-10, CXCL-6 and the factors NFκB, VEGF-A, TGF-β. Virus infection of human cells generally leads to activation of signalling pathways that cause the induction of pro-inflammatory cytokines and transcription factors (Mogensen, T. H., and S. R. Paludan, 2001 Molecular pathways in virus-induced cytokine production. Microbiology and Molecular Biology Reviews 65: 131-+). It was considered interesting, therefore, to analyse both the effect of HSV1716 infection at MOI 50 on MDM gene expression and differences between infections performed under normoxic and hypoxic conditions.
(84) HSV1716 infection at MOI 50 caused the induction of pro-inflammatory cytokines by 48 hours, and the increase in expression was especially observed under hypoxic conditions, whereas no considerable changes were observed in normoxia. Cytokines IL-8 and IL-1 were found to be 5- and 7-fold upregulated, respectively, in hypoxia. A 5-fold increased expression under hypoxic conditions was also observed for NFκB; however, surprisingly, NFκB is down-regulated by 5 folds in normoxia. This finding suggests a different response of MDM to HSV1716 which could have higher inflammatory properties in the absence of oxygen. If this is the case, this would suggest that HSV1716 acquires a greater viral potency in hypoxia: this result would further support the rationale of using virus delivery by MDM to target central areas of tumor, difficult to access through different ways.
(85) The anti-inflammatory cytokine IL-10 and the growth factor TGF-β are down-regulated by 5 folds after HSV1716 infection, but only in normoxic conditions. Indeed, under hypoxia, a 4-fold increase in the anti-inflammatory IL-10 expression was observed, possibly opposing the pro-inflammatory effect. Interestingly, there is strong up-regulation of VEGF-A under hypoxic conditions (21 fold), which could be partly due by the fact that VEGF-A is normally involved in the hypoxic response.
(86) In summary, this study demonstrates that HSV1716 induces tumor cell death in prostate and breast cancer cell lines, and is able to replicate in MDM and disseminate in the surrounding microenvironment. In addition, results show that when delivered through MDM, HSV1716 causes cell death in multicellular 3D spheroids; therefore, the macrophage-mediated delivery of oncolytic HSV1716 to tumors constitutes a possible therapeutic approach to treat solid tumors. The great safety profile of HSV1716, shown by previously performed clinical trials, makes the possibility of using it as a MDM deliverable therapy an exciting opportunity to further increase the range of treatments that can be offered to cancer patients.
References for Example 2
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Scott, 1953 THE CONCENTRATION OF OXYGEN DISSOLVED IN TISSUES AT THE TIME OF IRRADIATION AS A FACTOR IN RADIOTHERAPY. British Journal of Radiology 26: 638-648. Griffiths, L., K. Binley, S. Iqball, O. Kan, P. Maxwell et al., 2000 The macrophage—a novel system to deliver gene therapy to pathological hypoxia. Gene Therapy 7: 255-262. Grimshaw, M. J., S. Naylor and F. R. Balkwill, 2002 Endothelin-2 is a hypoxia-induced autocrine survival factor for breast tumor cells. Molecular Cancer Therapeutics 1: 1273-1281. Harris, A. L., 2002 Hypoxia—A key regulatory factor in tumor growth. Nature Reviews Cancer 2: 38-47. Harrow, S., V. Papanastassiou, J. Harland, R. Mabbs, R. Petty et al., 2004 HSV1716 injection into the brain adjacent to tumor following surgical resection of high-grade glioma: safety data and long-term survival. Gene Therapy 11: 1648-1658. He, K.-F., L. Zhang, C.-F. Huang, S.-R. Ma, Y.-F. Wang et al., 2014 CD163+ Tumor-Associated Macrophages Correlated with Poor Prognosis and Cancer Stem Cells in Oral Squamous Cell Carcinoma, BioMed research international 2014: 838632. He, W. S., X. F. Dai, M. Jin, C. W. Liu and J. H. Ren, 2012 Hypoxia-Induced Autophagy Confers Resistance of Breast Cancer Cells to Ionizing Radiation. Oncology Research 20: 251-258. Jiang, G., C. S. Yang, D. Xu, C. Sun, J. N. Zheng et al., 2014 Potent anti-tumor activity of a novel conditionally replicating adenovirus for melanoma via inhibition of migration and invasion. British Journal of Cancer 110: 2496-2505. Kacinski, B. M., 1995 CSF-1 AND ITS RECEPTOR IN OVARIAN, ENDOMETRIAL AND BREAST-CANCER. Annals of Medicine 27: 79-85. Kandel, J. J., D. J. Yamashiro and J. Kitajewski, 2011 Angiogenesis in Tumor Development and Metastasis, pp. 81-93 in Therapeutic Angiogenesis for Vascular Diseases: Molecular Mechanisms and Targeted Clinical Approaches for the Treatment of Angiogenic Disease, edited by M. Slevin. Springer-Verlag Berlin, Berlin. Kim, J., J. Y. Cho, J. H. Kim, K. C. Jung and C. O. Yun, 2002 Evaluation of E1B gene-attenuated replicating adenoviruses for cancer gene therapy. Cancer Gene Therapy 9: 725-736. Kim, S., S. W. Cho, H. S. Min, K. M. Kim, G. J. Yeom et al., 2013 The expression of tumor-associated macrophages in papillary thyroid carcinoma. Endocrinology and metabolism (Seoul, Korea) 28: 192-198. Koong, A. C., N. C. Denko, K. M. Hudson, C. Schindler, L. Swiersz et al., 2000 Candidate genes for the hypoxic tumor phenotype. Cancer Research 60: 883-887. Kurahara, H., S. Takao, T. Kuwahata, T. Nagai, Q. Ding et al., 2012 Clinical Significance of Folate Receptor beta-expressing Tumor-associated Macrophages in Pancreatic Cancer. Annals of Surgical Oncology 19: 2264-2271. Lan, C. Y., X. Huang, S. X. Lin, H. Q. Huang, Q. C. Cai et al., 2013 Expression of M2-Polarized Macrophages is Associated with Poor Prognosis for Advanced Epithelial Ovarian Cancer. Technology in Cancer Research & Treatment 12: 259-267. Lewis, J., R. J. Landers, R. D. Leek, K. Corke, A. L. Harris et al., 1997 Role of macrophages in tumor angiogenesis: Regulation by hypoxia. Journal of Pathology 182: A1-A1. Lewis, J. S., R. J. Landers, J. C. E. Underwood, A. L. Harris and C. E. Lewis, 2000 Expression of vascular endothelial growth factor by macrophages is up-regulated in poorly vascularized areas of breast carcinomas. Journal of Pathology 192: 150-158. Li, X., H. Kimura, K. Hirota, H. Sugimoto and H. Yoshida, 2005 Hypoxia reduces constitutive and TNF-alpha-induced expression of monocyte chemoattractant protein-1 in human proximal renal tubular cells. Biochemical and Biophysical Research Communications 335: 1026-1034. MacKie, R. M., B. Stewart and S. M. Brown, 2001 Intralesional injection of herpes simplex virus 1716 in metastatic melanoma. Lancet 357: 525-526. Martinez, F. O., S. Gordon, M. Locati and A. Mantovani, 2006 Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: New molecules and patterns of gene expression. Journal of Immunology 177: 7303-7311. Mogensen, T. H., and S. R. Paludan, 2001 Molecular pathways in virus-induced cytokine production. Microbiology and Molecular Biology Reviews 65: 131-+. Mukhtar, R. A., A. P. Moore, V. J. Tandon, O. Nseyo, P. Twomey et al., 2012 Elevated Levels of Proliferating and Recently Migrated Tumor-associated Macrophages Confer Increased Aggressiveness and Worse Outcomes in Breast Cancer. Annals of Surgical Oncology 19: 3979-3986.
Example 3
(88) Isolation and Culture of Human Macrophages
(89) Mononuclear cells were isolated from platelet-depleted buffy coats (Blood Transfusion Service, Sheffield, UK) using Ficoll-Paque Plus (Amersham Pharmacia, St. Albans, UK) and monocyte-derived macrophages (MDM) prepared as described previously.sup.21,22.
(90) Endothelial Cell Cultures
(91) Human Umbilical Vein Endothelial Cells (HUVEC) were seeded for 24 h onto collagen-coated (0.1 mg/ml, human type IV) membranes containing a 5 μM pore PET membrane (Neuroprobe).
(92) Human Multi-Cellular Tumor Spheroids
(93) Human prostate cancer cell line, LNCaP, were seeded (5×10.sup.3) in 100 ul medium into each well of a 2% agarose (Sigma, Dorset, UK) coated 96-well tissue culture plate. After 7-10 days, each well contained a tumor spheroid with an average diameter of 700-800 um.sup.21.
(94) Infection of Primary Macrophages
(95) Day 3 MDMs were infected with a replication deficient adenovirus (CMV-AdV5-GFP (driven by a CMV promoter). Virus optimization and GFP expression levels are described in.sup.21.
(96) Cellular Uptake of Magnetic Nanoparticles by Macrophages
(97) MDMs (infected with AdCMV-GFP) were cultured overnight with 100 ug/ml SPIOs (25 nm) (Sigma-Aldrich, Poole, UK). SPIO accumulation in cells was previously assessed by flow cytometry and confirmed by attraction of the cells towards a magnet placed at the side of the culture dish as observed by light microscopy as described in Muthana, M. et al. A novel magnetic approach to enhance the efficacy of cell-based gene therapies. Gene Ther (2008). Cell viability following SPIO uptake by macrophages was also measured and compared to cells that were not incubated with SPIOs using the DNA dye propidium iodide (PI). No statistically significant difference was observed between the two groups p=0.4 (
(98) In Vitro Trans-Endothelial Flow Assay
(99) The trans-endothelial migration (TEM) chamber was assembled as shown in (
(100) Spheroid infiltration by MDMs was then assessed using a fluorescent microscope to detect the GFP positive cells and flow cytometry using enzymatically-dispersed spheroids. To determine the iron content within SPIO-loaded macrophages, cell pellets were solubilized in 70% nitric acid for 7-14 days prior to analysis. Iron concentrations were quantified against a calibration standard iron solution (Fischer Scientific, Loughborough, UK) by Atomic Emission Spectroscopy using Varian Vista-M PX.sup.14.
(101) Orthotopic Prostate Xenograft Model
(102) Male CD1 athymic mice were used in these studies (Charles Rivers, UK). One million LNCaP:LUC cells (a kind gift from Professor Magnus Essand, Uppsala Sweden) were mixed 1:1 in Matrigel and injected into the dorsolateral prostate. Tumor size was determined by assessment using bioluminescent IVIS imaging and measuring the daily weights of the mice as described in in Muthana, M. et al. Macrophage Delivery of an Oncolytic Virus Abolishes Tumor Regrowth and Metastasis After Chemotherapy or Irradiation. Cancer Res, doi:0008-5472.CAN-12-3056 [pii]10.1158/0008-5472.CAN-12-3056 (2013). Tumor-bearing mice were used in experiments approximately 14 days following implantation or 21 days in the metastases model when the pulmonary tumors develop following implantation of the tumor cells into the prostate.sup.21.
(103) Use of the MRI Scanner to Direct Cell Movement
(104) Three million MDMs with or without SPIOs were administered via tail vein in 100 μl volume of PBS (n=5), control groups received 100 ul PBS (n=5), or 100 ul PBS containing 3×10.sup.6 macrophages without SPIOs (n=5). Immediately after MDM administration mice were anaesthetized with gaseous isoflurane and then secured within a magnet-compatible holding capsule and MR targeting was carried out immediately.
(105) Mice were split into 2 groups of n=5. Group 1 was a time-matched control without MR targeting and Group 2 underwent 1 hour of MR targeting (see above) with gradients selected for coarse steering to the tumor site for the Prostate (−z, −y). For steering to the lungs (+z and −y gradients), the absence of an x gradient should ensure even distribution of magnetic particles in each lung.
(106) The force on magnetically labeled cells is dependent on whether the SPIOs have become magnetically saturated. When unsaturated, the force is dependent on the magnetic susceptibility of the SPIOs, the magnetic field and also the magnetic field gradient (Pankhurst, Q. A., Connolly, J., Jones, S. K. & Dobson, J. Applications of magnetic 443″ nanoparticles in biomedicine. J Phys D Appl Phys 36, R167-R181, (2003)).
(107) However once the SPIOs reach saturation, the force is no longer dependent on the magnetic susceptibility of the particle but the saturation magnetization and as such only the magnetic field gradient will affect the force applied to the cells (Riegler, J. et al. Targeted magnetic delivery and tracking of cells using a magnetic resonance imaging system. Biomaterials 31, 5366-5371, (2010).) SPIOs typically reach magnetic saturation well below 1 T, for example in Riegler et al. 2013, where the SPIOs become saturated at around 300 mT, therefore MRT is feasible on clinical MRI systems provided the same magnetic field gradient is used ˜300 mT/m.
(108) Following MRI-steering, high-resolution RARE and FLASH images of the tumor (prostate only) were taken. Once complete relaxometry-using MSE and MGE was performed to assess the transverse relaxation rates. After treatment, animals were sacrificed and tissues including tumors, kidney, liver, lungs and spleen, were either paraffin wax embedded and fixed for immunohistochemistry or analyzed by flow cytometry to determine macrophage uptake.
(109) Endothelial Cell Cultures
(110) Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Promocell, (Heidelberg, Germany) and used in the experiments up to passage 8. Cells (150,000) were seeded for 24 h onto collagen-coated (0.1 mg/ml, human type IV) membranes containing a 5 μM pore PET membrane (Neuroprobe). This resulted in a confluent monolayer of HUVECs on filters as seen by CD31 staining (data not shown).
(111) Infection of Primary Macrophages
(112) Day 3 MDMs were infected with a replication deficient adenovirus (CMV-AdV5-GFP). The E1A/B-deleted adenoviral vectors, CMV-AdV5-GFP (driven by a CMV promoter) was isolated from a single plaque, expanded in 293 human embryonic kidney (HEK) cells All the viruses were purified by double caesium gradient centrifugation, and titered by plaque assay on 293 cells with the titer expressed as plaque forming units (PFU)/cell. Virus optimization and GFP expression levels in macrophages are described in.sup.21.
(113) Flow Cytometric Analysis
(114) Single cell suspensions were obtained by trypsinizing MDMs (including co-transduced MDMs). Cells were then incubated for 30 min at 4° C. with mouse anti-CD14, 1:100 in PBS containing 1% BSA (Sigma) to prevent non-specific antibody binding. Alternatively, spheroids were digested using 0.25% trypsin/EDTA to separate the tumor cells and infiltrated macrophages and cell death was analysed by flow cytometry by adding propidium iodide (Sigma) to the cells immediately before running on the flow cytometer.
(115) Cellular Uptake of Magnetic Nanoparticles by Macrophages
(116) For the nanoparticle cellular uptake studies, MDMs (infected with AdCMV-GFP) were cultured overnight with magnetic nanoparticles 100 μg/ml SPIOs (25 nm) (Sigma-Aldrich, Poole, UK). MNP accumulation in cells following incubation with SPIOs was assessed by flow cytometry, this included measuring cell viability with propidium iodide (PI) as described by us.sup.14 and confirmed by attraction of the cells towards a magnet placed at the side of the culture dish as observed by light microscopy (Leica Microsystems UK Ltd).
(117) HSV1716 Virotherapy
(118) For therapeutic studies LNCaPs or macrophages were infected with HSV1716GFP (an HSV1716 variant with a GFP expression cassette inserted in the deleted ICP34.5 loci) at a multiplicity of infection (MOI) of 5 or 50. Cell death was assessed by flow cytometry 96 h post infection using PI staining. Viral particles were detected in clarified supernatants of infected macrophages using a titration assay on Vero cells to determine plaque-forming units.
(119) Mice received tail vein injections of either 3 million MDM alone or armed with HSV1716 at MOI 50, 1×10.sup.7 pfu HSV1716 only or PBS (n=5 mice/group). Of note, 3 groups of mice were administered MDM+OV, one group underwent MRT for 1 h, one sat in the MRI scanner for 1 h but had no MRT (MDM+OV no MRT) and another group did not enter the MRI scanner (MDM+OV). Tumor size was monitored by IVIS Lumina II imaging (IVIS, Caliper Life Sciences). Animals were sacrificed once tumors reached the maximum volume permitted by UK Home Office Regulations, and 1 hour before sacrifice, mice were injected intravenously FITC:Lectin (used for detecting tumor vasculature). Of note, mice receiving PBS and MDM only were culled on day 14-post treatment due to large tumor size. All other tumors were removed on day 21. Excised tissues including tumors, kidney, liver, lungs, and spleen were embedded in OCT or paraffin wax for histologic labeling studies.
(120) Analysis
(121) Tissues were divided into two; one part was formalin fixed for immunohistological analysis and the other was dissected free of adherent fibrous and fatty tissue and treated with collagenase.
(122) Flow cytometry: cell viability was determined using LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen). All FACS data were analyzed on an LSR II flow cytometer (BD Biosciences) using FlowJo software (Tree Star).
(123) Histology: Five micron sections of all organs were incubated with specific antibodies for target antigens; for the vasculature we used CD31 (1:100), (AbD Serotec) and for macrophages human CD68 (Dako, Ely, UK) at 1:100 and to detect adenovirus we used E1A at 1:50 (Millipore, UK). A biotinylated secondary antibody system was used in conjunction with a streptavidin-conjugated HRP. Peroxidase activity was localised with diaminobenzidine (Vectastain Elite ABC kit, Vector Labs). To detect iron in the tumors (where cell densities were high) sections were stained with Perls Prussian blue and counter-stained with eosin for improved contrast. To detect cancer cells in the lungs all lung sections were stained with Epithelial cell adhesion molecule (EPCAM) or Hematoxylin and eosin (H&E). All immune-localization experiments were repeated on multiple tissue sections and included isotype-matched controls for determination of background staining.
(124) Statistical Analysis
(125) Data are means±SEM. Student's t test were used to analyze the statistical significance of the data. Differences were termed significant a P value of less than 0.05.
(126) Supplementary Methods
(127) Mouse procedures and human monocyte isolation were conducted in accordance with the University of Sheffield Ethics Committee and UK Home Office Regulations under the Animals (Scientific Procedures) Act 1986.
(128) Results
(129) We show that therapeutic cells armed with an oncolytic virus (HSV1716) can be magnetically labeled using super-paramagnetic iron oxide nanoparticles (SPIOs) and then steered from the bloodstream into deep target tissues (primary and secondary tumors) using pulsed magnetic-field gradients within a magnetic resonance imaging (MRI) system. Use of this technique resulted in a marked increase in cell delivery to tumors and a significant reduction in tumor burden and metastasis. Our study, therefore, shows that clinical MRI scanners could be used, not only to image such magnetically labelled cells after their injection into the body, but also to steer them specifically to one or more target sites within the body. We describe the use of magnetic resonance targeting (MRT) to increase delivery of macrophages to tumors.
(130) We show that it is possible to manipulate the spatial field gradient coils of the MRI scanner to shape the magnetic field in/around a tumor, thereby non-invasively steering magnetically labeled cells towards it (
(131) We previously showed that such MRT could be used to both image and move cells in an in vitro vascular bifurcation model (a 2D tube that mimics arterial bifurcation).sup.13. Here, we show that MRT can also be used to ‘steer’ magnetic macrophages in vivo—i.e. from the bloodstream into two target organs, orthotopic prostate tumors and their pulmonary metastasis in mice. We have used macrophages as an example of a cellular vehicle as these cells are highly phagocytic allowing them to readily consume SPIOs whilst retaining their magnetic properties.sup.14,18,19. Such bone marrow-derived cells are increasingly being used in cell-based therapies for such diseases as cancer.sup.20-22, infarcted myocardium.sup.23, spinal cord injury.sup.24, cerebral ischemia.sup.25, degenerative diseases like Parkinson's Disease.sup.26 and Alzheimer's Disease.sup.27.
(132) Before applying MRT techniques in vivo we first established that a pre-clinical 7 T MRI system fitted with a 600 mT/m gradient coil set could generate substantial actuation forces on magnetic macrophages in vitro by steering them across an endothelial layer into 3D human tumor spheroids (MTS). To do this, we designed a trans-endothelial migration (TEM) flow chamber in which human macrophages circulated across the surface of a perforated membrane coated with a layer of human vascular endothelial cells, thereby mimicking flow in tumor venules. MTS were cultured in a non-adherent chamber below the membrane (
(133) MRT experiments used a pulsed magnetic field gradient (2 ms on, 7 ms off, 50% strength ˜300 mT/m.sup.13) for 1 hour in the direction of the spheroids (
(134) We then investigated whether such an MRI gradient system could be used to steer magnetic macrophages to tumors in vivo (
(135) MRT significantly (p=0.0001) increased uptake of SPIO-loaded macrophages in primary prostate tumors (42.2%±2.5) compared to the control group (7.17%±0.8) (
(136) Additional controls included tumor-bearing mice: (i) with unlabelled macrophages and MR targeting, and (ii) with unlabelled macrophages without MR targeting. For these control groups, we detected very few macrophages within tumors as confirmed by MRI imaging (
(137) MRT has particular application when tumors are difficult or impossible to remove surgically, as in the lung, brain, liver or spinal cord. Separate MRT sessions could enable targeting of a cell-based therapy to one or more metastatic lesions in cancer patients. In a second in vivo experiment we steered magnetic macrophages into lungs containing micro-metastases in our tumor-bearing mice. MRT was again used to steer magnetic macrophages towards the lungs following administration of 3 million macrophages. Mice without application of MRT but exposed to the magnetic field of the scanner, for the same length of time were used as a time-matched control.
(138) Flow cytometric analysis of enzymatically dispersed lungs showed the presence of significantly more human CD14+ macrophages following MRT than in the control group (17.7%±4 vs. 4.4%±2.6, respectively) (
(139) In a final experiment to assess the therapeutic benefits of MRT we targeted SPIO-loaded macrophages armed with the therapeutic oncolytic virus (OV) HSV1716 to tumor bearing mice. HSV1716 replication is supported by PC3 prostate cancer cells {Conner and Braidwood, Cancer Gene Ther. 2012 July; 19(7):499-507} and here we show for the first time oncolysis in LNCaP cells in both hypoxic (0.5% O.sub.2) and normoxic (20% O.sub.2) conditions (
(140) Bioluminescence of mice receiving macrophage OV therapy with or without MR targeting on the first day of treatment (day 0) and at the end of the experiment (day 21) showed this marked reduction of the primary tumor (
(141) MR images of mice receiving macrophage OV therapy with or without MRT on the first day of treatment (day 0) and at the end of the experiment (day 21) reflect this marked reduction of the primary tumor. Interestingly, the tumors from mice treated with OV or MDM carrying OV were considerably paler and less vascularized and this correlated with a reduced microvessel density (MVD) compared with the PBS or MDM alone group. In mice undergoing MRT following macrophage-delivered OV significantly more necrosis (p<0.001) in tumors was observed than in the absence of MRT.
(142) We next determined how these therapies influenced the development of pulmonary metastases. Few metastases were detected in mice injected with PBS or MDM alone since primary tumors in these groups had to be removed by day 14 (due to their size). Therefore, it was not valid to compare metastases in these control groups with the other experimental groups. However, the formation of lung metastases was markedly reduced when mice received MRT following delivery of OV-bearing macrophages in comparison to when no MRT was used (
(143) In summary, we show that an MRI scanner can be used to non-invasively steer cells to both primary and secondary tumors within the body leading to a significant improvement in therapeutic outcome. Moreover, relaxometry measurements suggest that MRI post MRT can be used to assess the efficacy of this approach. Whilst this study has focused on cell delivery to tumors, the technology could be used to target any cells (e.g. stem cells) to a given tissue including non-phagocytic cell types which could be ‘magnetised’ using SPIO-conjugated antibodies directed against proteins on their cell surface.
(144) The use of magnetic resonance targeting, which exploits the magnetic field gradients within magnetic resonance imaging systems to increase delivery of cells, is ideally suited to deep or superficial tissue. The question of clinical translation is dependent on the ability to provide the same targeting force on a clinical MRI system. Clinical scanners, with high performance magnetic field gradient systems of 300 mT/m, are already in use and as such have the potential to produce similar forces. Moreover, we were able to image the cell distributions following MRT, indicating the possibility for real-time image-guided targeting using an MRI system. These findings support the potential value of MRT with concomitant imaging for improved targeting of cells for therapy.
References for Example 3
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