EVALUATING THE EFFICACY OF INTRAVESICAL LACTOBACILLUS RHAMNOSUS GG AS A NOVEL THERAPEUTIC APPROACH IN BBN MURINE MODEL OF BLADDER CANCER.

The current standard of care for high-risk non-muscle invasive bladder cancer (HR-NMIBC) is Bacillus Calmette Guerin (BCG), however, it carries a non-responder rate of 30-50% with high risk of progression and side effect profile. Sequential intravesical gemcitabine and docetaxel (Gem/Doce) is an increasingly utilized treatment for HR-NMIBC, although prospective validation is pending. Our previous analyses have demonstrated an increased abundance of Lactobacillus rhamnoses GG (LGG) within the tumor stroma of responders, thus, we aimed to assess the role of intravesical LGG instillation as potential therapy in tumor-bearing mice. We used the BBN model to recapitulate the histological and genetic characteristics of human bladder cancer in C57BL/6 mice. Upon ultrasound confirmation of tumor, mice were treated with live LGG (106 CFU) given via intravesical instillation for six weeks. Tumor burden was measured weekly with US. Additional comparative therapy included saline, BCG (106 CFU), and Gem/Doce. (Figure 1A) Cytokine urine analysis utilizing CodePlex Secretome Adaptive Panel* was collected at week 4. Immunohistochemistry was performed with bladder tissue and T-lymphocyte and total CD8 T-lymphocytes per high-power field were calculated. Following monocyte purification and stimulation, we assessed differentiation into macrophage and dendritic cells in both BCG and LGG group using FACS Aria, and processed results through Cell Quest. Additional supernatant cytokine production of monocytes, T-cells, and monocytes and T-cells to LGG and BCG was evaluated using Human CodePlex Adaptive Panel**. Complete response was seen in LGG (4/10), NS (0/10), BCG (2/10), and Gem/Doce (4/10) with tumor volume in LGG and Gem/Doce (5.1 cm3, 5.6 cm3). (Figure 1B). Week 4 urine cytokine demonstrated increased expression of KC (CXCL-1, 538.95 pg/ml), IP-10(CXCL10, 355.17 pg/ml), IL-6(477.05 pg/ml), IL-4(89.35 pg/ml), IL-1B (240.21 pg/ml) and MIP-1a (210.67 pg/ml) in LGG group (p<0.001).(Figure 1E) IHC of LGG demonstrated an increased Tumor/Stroma T-cell Infiltration;(0.93;±0.70).(Figure 1C, D) In vitro stimulation of human monocytes (CD14+/CD16+) and T-cells (CD3+/CD8+/CD4+) with LGG resulted in increased of IL-6 (1.82±;0.82pg/ml), IL-7 (0.68±0.14 pg/ml), and Granzyme B (0.64;±;1.91 pg/ml),;(Figure 1F) with increasing differentiation to immature dendritic cells (CD1+/CD11C-) (88.41 vs 54.33%) Analysis of a murine intravesical therapy model revealed superior tumor response in both LGG and Gemcitabine/Docetaxel groups compared to control NS and standard of care BCG in the treatment of BBN induced tumor. Analysis of urinary LGG cytokine profile demonstrates enhanced immune activation through dendritic cell differentiation and Th2 pathway with direct antitumor effects through enhanced apoptosis.**(GM-CSF, Granzyme B, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-1α, MIP-1β, Perforin, sCD137, TNF-α, TNF-β).*(GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17A, IP-10, KC, MCP-1, MIP-1α, RANTES, TNF-α) [ABSTRACT FROM AUTHOR]