Recombinant h IFN-α2b-BCG inhibits tumor growth in a mouse model of bladder cancer

Abstract

Abstract. Bacillus Calmette-Guérin (BCG) reduces the recurrence and progression of non-muscle invasive bladder cancer. The present study aimed to investigate the impact of a recombinant hIFN-α2b-secreting BCG (rBCG) on the mouse bladder MB49 cell line and an orthotopic mouse model of bladder cancer. MB49 cells were cultivated in the presence or absence of rBCG, BCG or BCG+hIFN-α2b. Cellular morphology and viability were assessed by microscopy and CCK-8 assay, respectively. Apoptosis was assessed by acridine orange, Hoechst 33258 staining and flow cytometry. MHC-I expression was assessed by flow cytometry. MB49 cells were transplanted into the bladders of C57BL/6 mice administered BCG, rBCG or BCG+hIFN-α2b. Local tissue Fas expression and T cell subsets were assessed by immunohistochemistry. Peripheral blood TNF-α and IL-12 levels were measured by ELISA, and circulating T lymphocyte subsets by flow cytometry. BCG, rBCG and BCG+hIFN-α2b increased the distortion and death of MB49 cells, yet rBCG reduced the proliferation and enhanced apoptosis most substantially. Apoptosis was increased after a 24-h co-culture with rBCG or BCG+hIFN-α2b. Mice administered rBCG survived longer than mice administered BCG (p<0.001), yet this result was not significantly different from mice administered BCG+hIFN-α2b. The average bladder weight was reduced by administration of rBCG (p<0.001). Fas expression and peripheral blood mTNF-α and mIL-12, cell counts of polymorphonuclear leukocytes, monocytes, T lymphocytes and CD4 + /CD8 + ratios were significantly increased by all BCG treatments (p≤0.05), yet monocyte and T lymphocyte counts were higher in mice administered rBCG than in mice treated with BCG or BCG+hIFN-α2b (p=0.000). These results indicate that in an orthotopic murine bladder cancer model rBCG possesses superior antitumor activity to BCG+hIFN-α2b.

Recurrent bladder cancer in aging societies: Importance of major histocompatibility complex class I antigen presentation

“…The expression of MHC class I genes is controlled by cytokines (eg, interferons) and various transcriptional factors and signaling pathways, including NF‐κB and NLRC5. In studies presented here, MHC class I levels were augmented on BC cells by BCG (good evidence), NDV (single study), cytokines (IFN‐α‐2b, single study), TLR3 (single study) and TLR7/8 agonists (single study) that improved anti‐BC immune responses 37,41,42,44,52 . BCG and NDV promoted the production of proinflammatory cytokines, including interferons 37,44 .…”

“…In studies presented here, MHC class I levels were augmented on BC cells by BCG (good evidence), NDV (single study), cytokines (IFN-α-2b, single study), TLR3 (single study) and TLR7/8 agonists (single study) that improved anti-BC immune responses. 37,41,42,44,52 BCG and NDV promoted the production of proinflammatory cytokines, including interferons. 37,44 Interferons induce antigen presentation by increasing the expression of MHC class I and the activity of the immunoproteasome.…”

“…Addition of hIFN‐α‐2b augmented MHC class I expression on MB49 cell line and prolonged mouse survival compared with BCG treatment only. All treatments reduced bladder size, but rBCG was superior to other treatments in immune cell recruitment into the tumor, resulting in augmented infiltration of monocytes/macrophages 41 . Ayari and colleagues employed MBT‐2 mouse model and a combination treatment with BCG and agonist of toll‐like receptor 3 (TLR3), the later known to induce secretion of proinflammatory cytokines by the immune cells.…”

Recombinant BCG to Enhance Its Immunomodulatory Activities

“…Therefore, treatment of these patients with an improved rBCG strain may potentially circumvent treatment failure. Intravesical instillation of rBCG secreting certain cytokines, such as IFN-γ [ 35 ], IFN-α [ 43 ] or IL-15 [ 57 ], or various chimeric proteins in which GM-CSF is fused to cancer antigens [ 67 , 68 , 69 ], has been shown to result in prolonged survival and enhanced tumor growth suppression compared to the installation of pBCG in various mouse models of bladder cancer. Expression of bacterial toxin fragments, such as PTA [ 93 ] or increased production of c-di-AMP [ 131 ], has also improved the performance of BCG against tumor growth in murine and models.…”

“…The direct effect of rBCG::hIFN-α on bladder cancer cells has been demonstrated by morphological changes in the MB49 cancer cell line, slower growth of these cells and induction of apoptosis upon co-culturing with rBCG::hIFN-α [ 43 ]. In a murine model of orthotopic bladder cancer, rBCG::hIFN-α induced bladder inflammation with strong infiltration of CD3 + lymphocytes, CD20 + monocytes and Gr1 + polymorphonuclear cells, and rBCG::hIFN-α-treated mice survived significantly longer than pBCG-treated mice.…”