Archives

  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br This project further examines cell death

    2019-10-21


    This project further examines cell death patterns of our novel chimeric vector and demonstrates evidence of robust ICD via caspase-independent pathways consistent with the vector’s parent orthopoxviruses.25,26 This has important implications for clinical translation as we move forward, given viral potential to activate other-wise immunosuppressive microenvironments, leading to an increase in the recruitment of immune cells to tumors and promotion of an anti-tumor immune response. Experiments outside of the scope of this work are planned to evaluate the relationship between cell death induced by this vector and anti-tumor immunity. 
    CF33-hNIS is tropic for tumor cells, and CF33-hNIS-infected tumor cells take up I-124 and are thus imageable using PET/CT. HCT116 and HT29-derived flank xeno-grafts were implanted in nude mice using 3 106 cells per tumor. When tumor volume reached a predetermined 150 mm3, mice received intratumoral (i.t.) injections of PBS, CF33-FLuc, or CF33-hNIS at a dose of 104 PFU. (A) Comparisons of PET/CT images on day 7 across treat-ment and control groups 2 h following i.v. injection of 200 mCi of I-124. (B) images of a single mouse over time. Note I-124 uptake in right-sided tumor on day 22 despite injection of only left-sided tumor. NIS, sodium iodide symporter; PFU, plaque-forming units.
    Our present study shows synergy between CF33-hNIS and I-131, which underscores the impor-tance of treatment strategies to augment and sustain viral efficacy as we move SB 203580 forward trans-lating these findings to clinical use. Several groups
    have demonstrated that the radiotracer uptake resulting from virally induced hNIS expression occurs in a dose-dependent manner regard-less of radiotracer and modality used (I-124PET, 99 mTcO4 SPECT, or I-123 scintigraphy). Herein, we have demonstrated robust radioio-
    dine uptake at viral doses 1–2 logs lower than previously reported, again speaking to the enhanced potency of CF33-hNIS.11,27–29 Our group and
    others have previously demonstrated that PET signal pattern also correlates with anti-tumor response to viral therapy.12 We have shown this with an earlier generation of this novel vector encoding a lucif-erase reporter gene and SB 203580 imaging,15 and have herein confirmed this principle using I-124 PET imaging. More extensive ex-periments are planned to confirm dosimetric delivery of radioisotope and viral tumor titration at each point of imaging.
    we have demonstrated enhanced potency over previous experi-ments.13,30 Previous studies from our group have shown radiotracer
    (I-124) uptake of engineered vaccinia virus GLV-1h153 in orthotopic triple-negative breast tumors in athymic nude mice at doses of 1 107 PFU and have demonstrated synergy with I-131 at dose of 1 106 PFU for triple-negative breast cancer and at 1 105 PFU for pancreas can-
    cer-derived xenografts, but at both of these doses of virus, little to no viral effect was seen on tumor treatment.13,31 We were pleased that im-
    ageable uptake and viral-induced tumoricidal activity occurred robustly with CF33-hNIS at doses of just 1 104 PFU, suggesting enhanced potency of our virus over prior constructs. Moreover, syn-ergy of CF33-hNIS with 2.0–2.5 mCi of i.v. I-131 is noted at similar i.v. doses as previously used with triple-negative breast models (5 mCi) and pancreas models, which demonstrated dose-dependent synergy at 1.0 and 2.5 mCi doses along with a vaccinia-based virus. Of note, Mansfield et al.32 have employed intraperitoneal dosing of
    www.moleculartherapy.org
    Figure 5. CF33-hNIS Kills Human Colon Cancer HCT116 Xenografts and Demonstrates Substantial Tumor Cell Damage in HT29 Xenografts
    Flank xenografts were created in nude mice with 5 106 cells. Tumors were injected with 1 105 PFU of CF33-NIS when they reached average volume of 150 mm3. (A and B) Results from HT29 (A) and HCT116 (B) xenografts are shown with virus (solid line) versus sham infection (dashed line). n = at least 4 per treatment group. (C) Representative immunohistochemical examination of intra-tumorally treated HT29 tumors 10 days following injection demonstrates abrogated cell division and substantial inflammation relative to controls: the tumor from the PBS-treated mouse (PBS-treated; left panels) is composed almost entirely of xenograft cells and contains only a small area of necrosis (n). Mitotic figures (lower left; arrows) are common in xenograft cells, and little, if any, inflammatory cell infiltrate is present. The xenograft tumor from CF33-hNIS-treated mouse (right panels) has an area of xenograft cells (top right; circled and labeled x) that is surrounded by a thick zone of inflammation (i). The photograph at bottom right shows small clusters of xenograft cells (x) surrounded by an abundant infiltrate of mononuclear (lymphocytes and macrophages) inflammatory cells. Mitotic figures are uncommon in xenograft cells. H&E stain is shown. (D) Representative immunohistochemical examination of intra-tumorally treated HCT116 tumors 10 days following injection demon-strates abrogated cell division and substantial inflammation relative to controls: the tumor from the PBS-treated mouse (left panels) has abundant areas composed of xenograft cells (x) intermixed with areas of necrosis (n). The empty spaces in the photograph (top left) are likely areas of liquefied, necrotic cell debris that were lost during tissue collection and processing. Little, if any, inflammatory cell infiltrate is present around or within this tumor. Mitotic figures are common in xenograft cells (lower left; arrows). The xenograft tumor from mouse 128_CF33-hNIS (right panels) has only a small area composed of xenograft cells (top right; circled and labeled x), large areas of necrosis (n), and thick, surrounding zone of inflammatory cell infiltrate (i). The photograph at bottom right shows the xenograft cells (below and right of dashed line) and the adjacent thick zone comprised of mononuclear (lymphocytes and macrophages) inflammatory cells (i). Xenograft cells in this tumor are smaller, and mitotic figures are uncommon. H&E stain is shown. NIS, sodium iodide symporter; NS, non-significant; PFU, plaque-forming units. *p < 0.05.