Neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical studies

Abstract

High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)-expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non-tumor-bearing and orthotopic glioma-bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

Fig. 1. In vitro characterization of the HB1.F3.CD NSC line

(A) Conventional cytogenetic analysis of HB1.F3.CD NSCs. (B) Tumor tropism of parental HB1.F3 and HB1.F3.CD NSCs in response to glioma cell-conditioned media at passages 21, 31 and 37 measured in vitro; mean ± SEM of triplicate measurements; where not visible, the error bars are contained within the graph. Data are expressed as % cells migrated, where 100% is 3 × 10⁴ (C) Flow cytometry analysis of HB1.F3.CD cells immunostained with anti-human nestin (blue line) or isotype control antibody (red line). ((D) E. coli cytosine deaminase expression in HB1.F3.CD NSCs (blue line); isotype control antibody was used as control (red line).

Fig. 2. HB1.F3.CD NSC tumor tropism in vivo in presence of dexamethasone or after radiation treatment

(A) Diagram of NSC injection (red box) caudal-lateral to tumor site (white box) or (F) into the hemisphere opposite the tumor site shown on 1x DAPI-stained representative brain tissue sections. Scale bar, 1 mm. (B–C, G–H) U251 tumors were established in the right frontal lobes of adult nude mice (n = 8). Mice received daily dexamethasone (0.2 mg/kg/day, injected intraperitoneally, started on day 7 and continued until mice were euthanized on day 14 or 17) 3 days prior to and 4 or 7 days after NSC injection. Cryosections of brains 4 days (B, G) and 7 days (C, H) after NSCs (labeled with CM Di-I, red; indicated with white arrows) were injected caudal-lateral to tumor (B, C) or into the hemisphere opposite to the tumor (G, H). Nuclei are indicated by DAPI (blue). Insets show high power images of cryosections.. (D, E) C57BL/6 mice were injected with 1 × 10⁵ syngeneic murine GL261 glioma cells into the frontal lobe. Seven days later, 1 × 10⁵ HB1.F3.CD NSCs (CM-DiI-labeled, red) were injected caudal-lateral to tumor. No immunosuppression was given. Representative DAPI-stained brain tumor sections from mice euthanized 4 (D) and 15 (E) days after NSC administration, showing glioma (dense blue nuclei) and HB1.F3.CD NSCs (red, indicated with white arrows). Scale bars, 100 μm. (I, J) Ten days after U251 tumors were established in the right frontal lobes of nude mice, 10 Gy irradiation was delivered to the brain. Ten days later, HB1.F3.CD NSCs were injected into the left hemisphere. Mice were euthanized 4 days after NSC injection. Shown are paraffin-embedded brain sections stained with a polyclonal antibody to CD/DAB-Ni to identify HB1.F3.CD NSCs (I, NSCs indicated with black arrows) or no primary antibody/DAB-Ni control (J).

Fig. 3. Safety of administering HB1.F3.CD NSCs into the brains of non-tumor-bearing nude mice

(A, B) Histological images of organs from non-tumor-bearing mice injected intracranially with 5 × 10⁵ HB1.F3.CD NSCs and euthanized 4 weeks (A) and 12 weeks (B) after injection of NSCs. (i–iii) H&E-stained brain tissue sections showing normal brain tissue with focal gliosis at NSC injection sites (arrows) (Ai, ii, iii and Bi, ii, iii. Scale bars Ai and Bi, 2000 μm). Representative sections from: (iv) heart, (v) lung, (vi) liver, (vii) spleen, (viii) pancreas, (ix) kidney/adrenal, (x) ovary, (xi) stomach, (xii) intestine, (xiii) skin. Scale bars, 100 μm.

Fig. 4. NSC-mediated enzyme/prodrug therapeutic paradigm and therapeutic effect

(A) Diagram of CD-expressing NSCs localized to tumor cells, and CD conversion of 5-FC to 5-FU, which readily diffuses out of the NSCs to selectively kill surrounding, dividing tumor cells. (B, C) H&E-stained brain tumor sections from U251 glioma-bearing mice that received HB1.F3.CD NSCs only (B) or HB1.F3.CD NSCs in combination with (C) treatment with 5-FC. White arrows indicate tumor region. (D, E) Tumor sections from U251 glioma-bearing mice that showed an exemplary response to treatment with NSCs and 5-FC 4 days after completion of one week of 5-FC treatment. Original tumor area is indicated by white dotted line and residual, apoptotic appearing tumor cells are indicated by green dotted line. Fluorescence immunohistochemistry with antibodies against Ki67 (D) and PCNA (E) revealed occasional residual tumor cell proliferation (green). NSCs (CM-DiI labeled, red) remaining after 5-FC treatment were not dividing, as determined by negative immunostaining for markers of cell division Ki67 and PCNA (white arrows, high-power image insets). Scale bars, 100 μm.

Fig. 5. Efficacy of HB1.F3.CD NSC + 5-FC combination treatment

(A) Survival curves for mice that received the indicated doses of NSCs (5 × 10⁴ and 1 × 10⁵ vs. no NSCs and 1 × 10⁴). P = 0.03. (B–E) Representative eGFP-DAB-Ni-stained brain tumor sections from mice treated with the combination of NSCs and 5-FC (group 3) (B, C) or NSCs only (group 6) (D, E). Tumor areas are outlined in red. Tumor section areas were multiplied by distance to next section through tumor to determine tumor volume. (F) Mean ln tumor volume and 95 % confidence limits of mice treated with the combination of HB1.F3.CD NSCs and 5-FC (groups 1–3) compared to control mice (groups 4–6, NSCs only; group 7, 5-FC only; group 8, tumor only) P = 0.041.

Fig. 6. HB1.F3.CD NSC safety/toxicity study data

(A) BUN and (B) GGT levels 90 days after tumor injection. (C, D). H& E-stained tissue sections from mice euthanized at day 30 that received (C) 1 × 10⁵ NSCs plus 5-FC (group 3) or (D) 1 × 10⁵ NSCs and no 5-FC (group 6). Images of: (i) heart, (ii) lung, (iii) liver, (iv) spleen, (v) pancreas, (vi) lumbar spinal cord (similar results for cranial, thoracic, and sacral spinal cord regions), (vii) kidney, (viii) adrenal, (ix) stomach, (x) intestine, (xi) skin, and (xii) testis (similar results for ovaries). (E–H) Images of brain tissue at 30 days after NSC injection; NSC injection site indicated by white arrows and tumor engraftment site indicated by black arrows. (E–G) Low and high power H& E-stained sections show a hypercellular focus (black arrow), consistent with tumor, measuring approximately 0.4 mm and located in the right frontal cortex, associated with hemosiderin pigment, gliosis and mild architectural distortion. Lateral to the tumor, 1.1 mm away, is a smaller focus of gliosis, consistent with the NSC injection site (white arrow), associated with hemosiderin pigment. (H) Brain tissue section immunohistochemically stained with an antibody to eGFP, indicating the main tumor mass and edges that infiltrate into the surrounding cortex up to 0.7 mm from the main mass, with preferential localization around blood vessels. Scale bars, 100 μm.