Concurrent Oncolysis and Neurolesion Repair by Dual Gene-Engineered hNSCs in an Experimental Model of Intraspinal Cord Glioblastoma
"> Figure 1
<p>In vitro evaluations of F3.hNSC and G55 cells for key biofactors related to NSC functional multipotency and GDEPT. (<b>A</b>) All cell lines showed a dose-dependent increase in VEGF production when incubated separately. F3.CD-TK cells had significantly lower VEGF secretion than F3.CD, G55, and HFB2050 in the largest culturing concentration (<span class="html-italic">p</span> < 0.05, one-way ANOVA with Tukey’s post hoc test; n = 9 wells/group). (<b>B</b>) Under a 1:1 ratio, the F3.CD-TK|G55 group expressed VEGF at a mean level that was statistically similar to that of G55 alone and significantly lower than F3.CD|G55, which had an average VEGF level significantly higher than the G55-only group (<span class="html-italic">p</span> < 0.05: * vs. G55+F3.CD or F3.CD-TK, # vs. all other groups; two-way ANOVA with Tukey’s post hoc test; n = 9 wells/group). (<b>C</b>,<b>D</b>) ELISA of BDNF production revealed that when cultured separately, F3.CD-TK and F3.CD both had a cell dose-dependent elevation of BDNF, but at the highest cell dose, F3.CD-TK had a significantly higher mean BDNF level than F3.CD. Notably, HFB2050 prototype hNSCs as positive control cells produced the highest amount of BDNF. Coculture of F3.CD-TK or F3.CD with G55 produced BDNF that, on average, was significantly higher than that of the F3.CD-TK, F3.CD, or G55 alone group (<span class="html-italic">p</span> < 0.05: * vs. G55, # vs. F3.CD; two-way ANOVA with Tukey’s post hoc test; n = 9 wells/group). (<b>E</b>) Culturing G55 cells alone for 96 h produced abundant Cx43 (grey bar) in either type of the 2-chamber system. Relative to the IRL of Cx43 in either pore size monoculture set as the reference value (100%), coculture of G55 with F3.CD-TK or F3.CD cells generated comparable group average IRLs of Cx43, which were, however, lower than G55 monoculture. Cocultured cells separated by smaller pores (ϕ = 0.4 µm) had significantly higher mean IRL values of Cx43 compared to that of the larger pore size (ϕ = 6 µm) group (<span class="html-italic">p</span> < 0.05: * vs. G55, # vs. 0.4 µm/ϕ group; one-way ANOVA with Tukey’s post hoc test; n = 9 wells/group). (<b>F</b>,<b>G</b>) Cocultured G55 and F3.CD-TK exhibited IR of Cx43 (green) alongside the membrane of the DiI+/red cells including loci within the pores (arrows; ϕ = 6 µm; <b>F</b>). The general IRL of Cx43 appeared to be stronger than that of Cx26, another gap junction protein expressed in the cell membrane (green IR in <b>G</b>) (arrowhead: loci in the pores; ϕ = 6 µm), which was assessed separately (scale bar: 10 µm). (<b>H</b>–<b>K</b>) Compared to the 96 h cell culture data, monoculture of G55 for 24 h had very low expression of Cx43 (insets in <b>H</b>): confocal z-stacks) and Cx26 (insets in <b>J</b>): confocal z-stacks). Both Cx43 and Cx26 expressions were markedly increased between G55 and F3.CD-TK cells after 24 h coculturing (Cx43: insets in <b>I</b>; Cx26: insets in <b>K</b>; scale bars for <b>H</b>–<b>K</b> insets: 20 µm). F3.CD-TK cells (with high hNestin IRL: red) also formed Cx43- or Cx26-containing gap junctions (color code: white) with G55 cells that were CD133+ (green; i.e., cells showing yellowish overlapping pixels in <b>I</b>,<b>K</b>; scale bars: 25 µm/<b>H</b>–<b>K</b>).</p> "> Figure 2
<p>In vitro assessments of the oncolytic effect of F3.hNSC treatment against G55 cells. (<b>A</b>) Three days after adding 5FC (2.1 mM) to F3.CD (DiI+, upper panel) and G55 coculture, G55 cells (DiI−) were physically attached, although ~60% of the cells had immunostains for cleaved caspase-3, a sign of apoptosis (lower panel); (<b>B</b>), DiI+ F3.CD cells surrounded by caspase-3+ G55 cells). (<b>C</b>) Orthogonal optical slicing confirmed the proper IHC signal loci of hNuclei (red) and capspase-3 (green) in z-stack images of G55 cells (DiI−). (<b>D</b>) In contrast, 3 days after delivering 5-FC (2.1 mM) and GCV (12 µM), F3.CD-TK+G55 coculture had a large fraction (~60–70%) of G55 cells that had died and fallen off, with most residual attached G55 cells being cleaved caspase-3+. F3.CD-TK cells spread more widely (in <b>D</b>) than F3.CD cells that often appeared in small clusters (in <b>B</b>) in contact with G55 cells (<b>B</b>,<b>D</b>: 30 µm/scale bar; arrows, putative G55 chemoattractant diffusion direction to induce F3.hNSC migration). (<b>E</b>) F3.CD-TK regimen treatment dose-dependently killed G55 cells. GCV was more potent than 5FC when given individually, and 5FC+GCV was markedly more effective than 5FC or GCV administered alone. Treatment of 2-APB, a connexin channel antagonist, dose-dependently blocked the G55 cytotoxic effect of 40 µM GCV (inset in <b>E</b>), suggesting that the effect is GJIC dependent. (<b>F</b>) Regarding the oncolytic efficacy difference, F3.CD-TK cells were significantly more potent than F3.CD cells when only 5FC was given for 72 h. (<b>G</b>) F3.CD-TK had a significantly stronger oncolytic effect compared to F3.CD when 5FC and GCV were both applied for 72 h. (<b>H</b>) A similar degree of efficacy difference was observed in the F3.CD-TK+G55 coculture treated with the same prodrug dosages for 36 h, and the formula of 5FC+GCV exposure for 72 h had the strongest oncolytic effect (n = 9 wells/group; * <span class="html-italic">p</span> < 0.05, one-way ANOVA with post hoc unpaired Student’s <span class="html-italic">t</span>-test).</p> "> Figure 3
<p>The effect of F3.CD-TK regimen on the motosensory function and overall survival of C6-ISCG animals. (<b>A</b>) C6-ISCG growth aggressively triggered the onset of the BBB score of 9 (i.e., the primary criterion of animal termination) as early as 16 days after G55 injection in the control animals that received F3.CD-TK+F3.CD cell debris+5FC&GCV. Rats treated with the F3.CD-TK regimen had significantly reduced hindlimb deficits starting ~2 weeks following the treatment compared to the F3.CD formula or control cell debris group (* <span class="html-italic">p</span> < 0.05, n = 6/group; two-way repeated measures ANOVA with Tukey’s post hoc test). (<b>B</b>) Kaplan–Meier curve data showed that overall survival in the F3.CD-TK-treated group was significantly longer than in the other two groups (two-sided <span class="html-italic">p</span> = 0.01, rank test and the test based on medians). The F3.CD formula did not significantly increase the survival of C6-ISCG rats relative to the control group (two-sided <span class="html-italic">p</span> > 0.05, the rank test and the test based on medians). (<b>C</b>) The F3.CD-TK regimen significantly improved mean bodyweight compared to F3.CD and cell debris groups (* <span class="html-italic">p</span> < 0.05 vs. F3.CD and cell debris, #<span class="html-italic">p</span> < 0.05 vs. 3 d after G55; two-way repeated measures ANOVA with post hoc Tukey’s test). (<b>D</b>) Animals on the F3.CD-TK regimen had significantly better forelimb locomotor function relative to the other two groups (<span class="html-italic">p</span> < 0.05: # vs. before surgery, * vs. cell debris or F3.CD; two-way repeated measures ANOVA with post hoc Tukey’s test). (<b>E</b>) Von Frey filament test revealed that rats in all three groups had a transient early phase mechanical hypersensitivity/allodynia, which was followed by a gradual development of mechanical hyposensitivity in the forepaw. The F3.CD-TK-treated animals had no discernible forepaw sensory abnormality for ~2 weeks of prodrug dosing, in contrast to the significantly perturbated sensory function shown in the other two groups. F3.CD-TK-treated animals had significantly less forepaw mechanical hyposensitivity than the other two groups during the terminal stage (<span class="html-italic">p</span> < 0.05: # vs. before surgery, * vs. F3.CD or cell debris; two-way repeated measures ANOVA with post hoc Tukey’s test).</p> "> Figure 4
<p>The effect of F3.CD-TK regimen on the autonomic function of C6-ISCG rats. (<b>A</b>) A noninvasive blood pressure monitoring system was used (VPR: volume pressure recording). (<b>B</b>,<b>C</b>) Three days succeeding C6 injection of G55 cells, all animals had significantly elevated systolic blood pressure, which returned to the pre-tumor level 3–7 days after F3.hNSC administration. Three weeks following C6 tumor seeding, the control group receiving cell debris had significant systolic and diastolic blood pressure decreases compared to baseline values and those of the two treated groups. (<b>D</b>) The changes resulted in significant reductions in mean artery pressure (MAP), which were efficaciously corrected by the F3.CD-TK and F3.CD treatments (<span class="html-italic">p</span> < 0.05: # vs. before surgery, * vs. cell debris or F3.CD; two-way repeated measures ANOVA with post hoc Tukey’s test). (<b>E</b>) Plethysmographic recording of conscious and free moving animals (upper panel) showed that in the termination week, the respiratory flow (RF: mL/s) pattern of the control rats was evidently abnormal (lower panel). (<b>F</b>) Additionally, the cell debris or F3.CD groups had significantly decreased respiratory rates (<span class="html-italic">f</span>: breaths/min) in weeks 1 and 2 after receiving intervention compared to pre-tumor baseline values. F3.CD-TK regimen, not F3.CD formula, maintained <span class="html-italic">f</span> within a normal range during that same period. (<b>G</b>,<b>H</b>) The reduced respiratory rate was caused by a significant increase of mean inspiration time (Ti: s) in the control group (<b>G</b>), while the mean expiratory time (Te: s) was comparable between the 3 groups (<b>H</b>). (<b>I</b>,<b>J</b>) The F3.CD-TK-treated group demonstrated significantly improved tidal volume (Vt: mL/per breath) (<b>I</b>) and minute ventilation (Ve: mL/min) (<b>J</b>) relative to the other two groups. Finally, no significant differences in the groups’ mean Vt and Ve were found between the 3 groups at the terminal stage (<span class="html-italic">p</span> < 0.05: # vs. before surgery, * vs. cell debris or F3.CD; two-way repeated measures ANOVA with post hoc Tukey’s test).</p> "> Figure 5
<p>The effect of F3.CD-TK regimen on the volume, histopathological feature, and apoptosis of C6-ISCG. (<b>A</b>–<b>C</b>) Termination time gross pathology photos showed that all spinal cords had intramedullary GBs in similar sizes (i.e., the dotted areas). (<b>D</b>–<b>F</b>) A similar outcome was found in transverse histopathology images (insets: left/H&E stain, middle/fluorescent confocal z-stack, and right/camera lucida image). The F3.CD-TK-treated tissue had a much wider intratumor distribution of DiI+ F3.CD-TK cells (middle inset: F3.CD-TK/<b>D</b> vs. F3.CD/<b>E</b> and cell debris/<b>F</b> as negative control). Regarding tumor topology, relative to F3.CD- or cell debris-treated tumors, post-F3.CD-TK regimen tumors had much denser and deeper longitudinal grooves and circumferential indentations, which increased the overall surface terrain (insets: <b>D</b> vs. <b>E</b>,<b>F</b>) to produce the largest surface area (S) to volume (V) ratio (see below). (<b>G</b>) Quantification of 3D tumor reconstruction data (samples in <b>D</b>–<b>F</b>) demonstrated that the mean GB volumes in the terminal stage were statistically indiscernible between the three groups. (<b>H</b>) The F3.CD-TK regimen more effectively impeded tumor growth, as per data in the scatterplot. (<b>I</b>) The mean S/V ratio of the F3.CD-TK-treated GBs was significantly higher than the other two groups (<span class="html-italic">p</span> < 0.05: * vs. cell debris, ∧ vs. F3.CD; one-way ANOVA with Tukey’s post hoc test). (<b>J</b>) The average percentage of G55 cell apoptosis in F3.CD-TK and F3.CD groups was significantly higher than that of the cell debris group: F3.CD-TK-treated animals had the highest rate of activated caspase 3+ G55 cells (confocal images in the left panel; statistics in the right panel; <span class="html-italic">p</span> < 0.05; one-way ANOVA with Tukey’s post hoc test; n = 6 rats/group). Scale bar: 1 mm/<b>A</b>–<b>F</b>.</p> "> Figure 6
<p>The impact of F3.CD-TK regimen on host neurite rescuing as well as neuroinflammation and neuroimmune modulation. (<b>A</b>–<b>C</b>) There was a significantly augmented group average IRL of neurofilament H (NF-H) in the host neurites (DiI−) of F3.CD-TK-treated C7 dorsolateral spinal cords within the grooved/indented interface around Rexed lamina-I (RL-I) compared to cell debris controls (n = 6/group; * <span class="html-italic">p</span> < 0.01, Student’s <span class="html-italic">t</span>-test). (<b>D</b>) About 10–15% NF-H+ (upper inset) neurites expressed the growth-associated protein 43-kD (GAP-43) that is a marker of neurite growth (arrows in (<b>D</b>); bottom inset: tissue location of the images). (<b>E</b>) Confocal imaging disclosed that ~6–10% NF-H+ axons within the interface zone were ensheathed by a thin layer of myelin basic protein (MBP) in F3.CD-TK-treated tissue only. (<b>F</b>) A similar fraction (i.e., 2–10%) of the NF-H+ neurites expressed vesicular glutamate transporter 1 (vGlut 1), an instrumental molecule of the propriospinal fiber terminals. (<b>G</b>–<b>I</b>) A significantly increased GFAP IRL presented along the interface zone and mainly on the host parenchyma side (left to the dotted line; upper inset: area sampled; <b>G</b>) in F3.CD-TK-regimen-treated spinal cords relative to controls (inset: area sampled; <b>H</b>) receiving cell debris (n = 6/group; * <span class="html-italic">p</span> < 0.01, Student’s <span class="html-italic">t</span>-test; <b>I</b>). Notably, cells with augmented GFAP expression contained no DiI (<b>G</b>), indicating that they were host astrocytes. DiI+ F3.CD-TK cells in the interface zone of an adjacent tissue section (dotted line in <b>G</b> lower inset) formed Cx43 gap junctions with both host cells (left side) and G55 cells (right side), which were revealed by yellow IR signals pointed by arrows in <b>G</b> lower inset. (<b>J</b>–<b>L</b>) Triple immunostaining disclosed that the F3.CD-TK regimen significantly increased the alternatively activated (arginase+/CD68+ and anti-inflammatory/immune modulatory) M2 microglia (arrowheads, <b>J</b>) quantity but decreased classically activated (CD86+/CD68+ and pro-inflammatory/cytotoxic) M1 (arrows, <b>J</b>) numbers when compared to the cell debris control formula (M2/arrowhead; M1/arrow in <b>K</b>), reducing the M1/M2 ratio (<b>L</b>; n = 6/group; * <span class="html-italic">p</span> < 0.01, Student’s <span class="html-italic">t</span>-test). The M2 increase in F3.CD-TK-treated tissues mostly occurred at loci deeper inside the host spinal cord (i.e., the upper left section in <b>J</b>) while the M1 cells were concentrated in areas adjacent to the tumor interface (i.e., the lower right section in <b>J</b>). In contrast, M1 microglia distribution was more even in the control tissue (<b>K</b>). Scale bars: 250 µm/<b>A</b>,<b>B</b>, and insets; 125 µm/<b>D</b> and insets; 10 µm/<b>E</b>; 20 µm/<b>F</b> and insets; 100 µm/<b>G</b>,<b>H</b>; and 80 µm/<b>J</b>,<b>K</b>, and insets.</p> "> Figure 7
<p>Comparison of self-clearance rate between F3.CD-TK and F3.CD cells in vivo. (<b>A</b>) The mean number of DiI+ cells was significantly less in F3.CD-TK-treated animals (upper panel) than F3.CD group (lower panel). (<b>B</b>) The residual F3.CD-TK cell number, averaged from DiI+ cell numbers from 6 slices (section thickness: 20 µm), with each sampled from one consecutive 500 μm-long tissue block, was merely ~25% of that of F3.CD-treated tissues (* <span class="html-italic">p</span> < 0.05, Student’s <span class="html-italic">t</span>-test). (<b>C</b>–<b>F)</b> Only ~5% of these residual F3.CD-TK cells (DiI+, <b>C</b>) expressed Ki67, a cell proliferation marker in the nucleus (<b>D</b>) and even fewer of which (~4%) had nuclear presence of cleaved capase-3 (<b>E</b>; DAPI nuclear stain in <b>F</b>), suggesting that they were in senescence after the vast majority had died off (statistics in <b>K</b>). (<b>G</b>–<b>J</b>) In contrast, ~72% of the residual F3.CD cells (<b>G</b>) exhibited nuclear IR of Ki67 (i.e., proliferating, <b>H</b>) and cleaved caspase-3 (i.e., experiencing apoptosis; <b>I</b>; DAPI nuclear stain in <b>J</b>; statistics in <b>L</b>). The F3.CD-TK/5FC+GCV regimen therapy more effectively eliminated proliferating donor cells. Scale bars: 700 µm/<b>A</b>; 40 µm/<b>C</b>–<b>J</b>.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. DiI Labeling of F3.CD and F3.CD-TK
2.3. In Vitro Assay for the Tumoricidal Effect of F3.CD and F3.CD-TK Formulas
- The dose–response oncolytic effect of 5FC on G55 converted by F3.CD or F3.CD-TK: G55 cells (1.5 × 104/well) and F3.CD or F3.CD-TK cells (0.75 × 104/well) were sequentially seeded in 24-well plates (BD Falcon; BD Biosciences, San Jose, CA, USA). After 24 h, a bolus dose of 5FC (0, 0.7 mM, 2.1 mM, or 7 mM) (Sigma-Aldrich) was added into the culture medium, and the culture was kept for another 72 h (total: 96 h).
- The dose–response effect of 5FC and GCV on G55 cells converted by F3.CD-TK cells: G55 cells (1.5 × 104/well) and F3.CD-TK cells (0.75 × 104/well) were sequentially seeded in 24-well plates (n = 5/group). After 24 h, a combinatorial dose of 5-FC (0, 0.7 mM, 2.1 mM, or 7 mM) and GCV (0, 4 µM, 12 µM, or 40 µM) (Sigma-Aldrich) was applied to the culture medium, and the culture was maintained for another 72 h (total: 96 h).
- Comparison of the oncolytic potency of individually or combinatorially applied 5FC and GCV in F3.CD-TK and G55 cocultures: G55 cells (1.5 × 104 per well) and F3.CD-TK cells (0.75 × 104 per well) were sequentially seeded in 24-well plates. After 24 h, a single dose of 5FC at 2.1 mM or GCV at 12 µM, or a combination of 2.1 mM 5FC + 12 µM GCV (i.e., optimal single doses determined in Steps i and ii), was applied to the culture medium to continue incubation for an additional 72 h.
- Evaluation of the oncolytic effect of consecutive prodrug administration on G55 cells converted by F3.CD-TK cells: G55 cells (1.5 × 104/well) and F3.CD-TK cells (0.75 × 104/well) were sequentially seeded in 24-well plates. After 24 h, 5FC or GCV was administered according to the following formulas: (1) 2.1 mM 5FC for 72 h; (2) 12 µM GCV for 72 h; (3) 2.1 mM 5FC for the first 36 h followed with 12 µM GCV for another 36 h; (4) 2.1 mM 5FC for the first 24 h with subsequent medium change with no precursor drug for 24 h (i.e., a washout period), followed by another 24 h treatment of 12 µM GCV; (5) 12 µM GCV for the first 36 h followed with 2.1 mM 5FC for another 36 h; (6) 12 µM GCV for 24 h with a subsequent 24 h washout period (see above), followed by another 24 h treatment of 2.1 mM 5FC; (7) combinatorial treatment of 2.1 mM 5FC and 12 µM GCV for 72 h; (8) no prodrug culturing for 72 h. At the end of each experiment, cell counting was conducted using the standard trypan blue method [5]. Each independent assay was performed in triplicate.
2.4. Immunocytochemical (ICC) Analysis of Activated Caspase 3
2.5. Enzyme-Linked Immunosorbent Assay (ELISA) of VEGF and BDNF
2.6. In Vivo Study Design
2.7. Intramedullary Spinal Cord Tumor Model
2.8. Administration of F3.hNSCs and Prodrugs
2.9. Functional Evaluation and Termination Criteria
2.10. Histopathological and Immunohistochemical (IHC) Procedures
2.11. Image Analysis
2.12. Statistical Analysis
3. Results
3.1. Expressions of VEGF and BDNF by F3.CD-TK and F3.CD Cells In Vitro
3.2. Connexins Expression and Gap Junction Formation by F3.CD-TK, F3.CD, and G55 Cells In Vitro
3.3. A stronger Cytotoxic Effect of F3.CD-TK (vs. F3.CD) Regimen on G55 Cells and Its Dependence on Gap Junction Channels
3.4. F3.CD-TK Regimen Robustly Improved Hindlimb Locomotion and Overall Survival of C6-ISCG Animals
3.5. F3.CD-TK Regimen Markedly Enhanced Wellbeing and Forelimb Motosensory Function of C6-ISCG Animals
3.6. Autonomic Improvements in C6-ISCG Animals following the F3.CD-TK Therapy
3.7. The Robust Effect of F3.CD-TK Regimen on Tumor Growth in C6-ISCG Animals
3.8. The Modifying Effect of F3.CD-TK Regimen on the Histopathological Feature of C6-ISCG
3.9. The Effect of F3.CD-TK Regimen on Inducing Apoptosis of C6-ISCG Cells
3.10. F3.CD-TK Regimen Rescued Host Neurites within the Interface Space between the Tumor and Spinal Cord
3.11. F3.CD-TK Treatment Improved Intraparenchymal Microenvironment via Neuroinflammation/Neuroimmune Modulation and GJIC Formation
3.12. The In Vivo Self-Clearance Rate Difference between F3.CD-TK and F3.CD Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Zeng, X.; Ropper, A.E.; Aljuboori, Z.; Yu, D.; Teng, T.W.; Kabatas, S.; Usuga, E.; Anderson, J.E.; Teng, Y.D. Concurrent Oncolysis and Neurolesion Repair by Dual Gene-Engineered hNSCs in an Experimental Model of Intraspinal Cord Glioblastoma. Cells 2024, 13, 1522. https://doi.org/10.3390/cells13181522
Zeng X, Ropper AE, Aljuboori Z, Yu D, Teng TW, Kabatas S, Usuga E, Anderson JE, Teng YD. Concurrent Oncolysis and Neurolesion Repair by Dual Gene-Engineered hNSCs in an Experimental Model of Intraspinal Cord Glioblastoma. Cells. 2024; 13(18):1522. https://doi.org/10.3390/cells13181522
Chicago/Turabian StyleZeng, Xiang, Alexander E. Ropper, Zaid Aljuboori, Dou Yu, Theodore W. Teng, Serdar Kabatas, Esteban Usuga, Jamie E. Anderson, and Yang D. Teng. 2024. "Concurrent Oncolysis and Neurolesion Repair by Dual Gene-Engineered hNSCs in an Experimental Model of Intraspinal Cord Glioblastoma" Cells 13, no. 18: 1522. https://doi.org/10.3390/cells13181522