Mesenchymal Stromal Cells Engineered To Express IL-12 for Breast Cancer Cellular Immunotherapy.

Abstract Interleukin 12 (IL-12), produced mostly by antigen presenting cells, is a heterodimeric Th1 cytokine which has been shown to induce a cellular immune response, cancer cell apoptosis, as well as anti-angiogenesis. Many studies have demonstrated the anti-cancer effectiveness of IL-12. However, significant toxicity from recombinant human IL-12 administration has been noted in phase I/II studies in advanced cancers. Therefore, sustained delivery of IL-12, avoiding toxic peaks seen with intermittent IV infusions, would be desirable. One means of delivering sustained and optimal amounts of IL-12 in cancer patients may be through a gene-enhanced cell therapy approach which our study assessed in a pre-clinical model of breast cancer. More specifically, we tested the use of an injectable subcutaneous implant comprising bone marrow-derived mesenchymal stromal cells (MSCs) gene-modified ex vivo to secrete IL-12 in mice with 4T1 breast cancer. Balb/c mice injected subcutaneously with the syngeneic 4T1 breast cancer cells at 2.5 x 104 cells/mouse received the following day, at the same location, Matrigel-embedded IL-12 gene-modified Balb/c-derived MSCs (IL-12 MSCs) or Matrigel-embedded control-vector modified MSCs (Control MSCs) at 106 cells/mouse. Tumor growth over time was determined in these two groups of mice, as well as in mice that received 4T1 cells only without any implants of MSCs (n=5-9/group). Our results revealed a substantial slowing of tumor progression in mice implanted with IL-12 MSCs as indicated by 100% of IL-12 mice being tumor-free on day 20 post-4T1 cell implantation, versus 0% of mice in both control groups, and with over 50% of IL-12 mice remaining tumor-free for over 50 days. Repeat experiments showed similar results. Analysis of plasma samples demonstrated significantly elevated levels of IL-12 and of interferon-γ in mice that received the implants of IL-12 MSCs, versus mice that received the Control MSCs. However, when tested in immunodeficient NOD-SCID mice, our approach revealed 0% tumor-free mice at day 20 post-implantation for all three groups, thus indicating that the anti-cancer effect seen in normal mice was mediated by bystander immune cells. In addition, we determined that the slowing of tumor growth was not due to systemic but to local delivery of IL-12, since IL-12 MSCs when injected in the flank contralateral to the 4T1 cells did not lead to any beneficial effect, all mice developing tumors similarly within 20 days. In an implant analysis experiment where 4T1 cells were admixed with MSCs in Matrigel and retrieved 2 weeks post-implantation, histopathology revealed substantially less tumor cells in implants with IL-12 MSCs as well as the presence of necrotic capillaries and necrotic tumor islets, in contrast to controls where most of the implant was occupied by tumor cells and with the presence of viable capillaries and tumor islets. Furthermore, we noted the beneficial effect of our gene-enhanced cell therapy strategy in the B16 mouse melanoma model as well. In conclusion, our investigation demonstrates the potential of employing autologous MSCs genetically engineered to secrete a therapeutic protein of interest, in this case IL-12, as part of a cell-based immunotherapy strategy for cancer therapy.

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