Preclinical data shows that intravesical instillation of Ty21a/Vivotif®, a commercial vaccine against typhoid fever, is an effective alternative option to standard Bacillus-Calmette-Guérin (BCG) immunotherapy for nonmuscle-invasive bladder cancer (NMIBC). Here we characterized the inflammatory effects of Ty21a on the bladder and investigated the immune mechanisms underlying tumor-regression towards the use of this bacterial vaccine in NMIBC patients. MB49 bladder tumor-bearing mice had significantly improved survival after intravesical instillations of Ty21a doses of 10 6 to 10 8 colony-forming units. By immunohistochemistry and morphology, both BCG and Ty21a instillations were associated with bladder inflammation, which was decreased with the use of low, but ,effective doses of Ty21a. Flow cytometry analysis showed a significant infiltration of T cells, natural killer (NK) cells, and myeloid cells, compared with controls, after a single dose of Ty21a, whereas this was only observed after multiple doses of BCG. The induced myeloid cells were predominantly neutrophils and Ly6C + CD103 + dendritic cells (DC), the latter being significantly more numerous after instillation of Ty21a than BCG. Ex vivo infection of human leukocytes with Ty21a, but not BCG, similarly significantly increased DC frequency. CD4 + and CD8 + T cells, but not NK cells nor neutrophils, were required for effective bladder tumor regression upon Ty21a treatment. Thus, the generation of antitumor adaptive immunity was identified as a key process underlying Ty21a-mediated treatment efficacy. Altogether, these results demonstrate mechanisms behind intravesical Ty21a therapy and suggest its potential as a safe and effective treatment for NMIBC patients.
The sewage sludge isolate Pseudomonas nitroreducens HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing revealed three circular DNA replicons: a chromosome and two plasmids. Plasmids were shown to code for putative adaptive functions such as heavy metal resistance, but with unclarified ability for self-transfer. About one-tenth of strain HBP-1′s chromosomal genes are likely of recent horizontal influx, being part of genomic islands, prophages and integrative and conjugative elements (ICEs). P. nitroreducens carries two large ICEs with different functional specialization, but with homologous core structures to the well-known ICEclc of Pseudomonas knackmussii B13. The variable regions of ICEPni1 (96 kb) code for, among others, heavy metal resistances and formaldehyde detoxification, whereas those of ICEPni2 (171 kb) encodes complete meta-cleavage pathways for catabolism of 2-hydroxybiphenyl and salicylate, a protocatechuate pathway and peripheral enzymes for 4-hydroxybenzoate, ferulate, vanillin and vanillate transformation. Both ICEs transferred at frequencies of 10−6–10−8 per P. nitroreducens HBP-1 donor into Pseudomonas putida, where they integrated site specifically into tRNAGly-gene targets, as expected. Our study highlights the underlying determinants and mechanisms driving dissemination of adaptive properties allowing bacterial strains to cope with polluted environments.
Bacillus Calmette-Guérin (BCG) instillations for the treatment of non-muscle-invasive bladder cancer patients can result in significant side effects and treatment failure. Immune checkpoint blockade and/or decreasing tumor-infiltrating myeloid suppressor cells may be alternative or complementary treatments. Here, we have characterized immune cell infiltration and chemoattractant molecules in mouse orthotopic MB49 bladder tumors. Our data show a 100-fold increase in CD45+ immune cells from day 5 to day 9 tumors including T cells and mainly myeloid cells. Both monocytic myeloid-derived suppressor-cells (M-MDSC) and polymorphonuclear (PMN)-MDSC were strongly increased in day 9 tumors, with PMN-MDSC representing ca. 70% of the myeloid cells in day 12 tumors, while tumor associated macrophages (TAM) were only modestly increased. The kinetic of PD-L1 tumor expression correlated with published data from patients with PD-L1 expressing bladder tumors and with efficacy of anti-PD-1 treatment, further validating the orthotopic MB49 bladder-tumor model as suitable for designing novel therapeutic strategies. Comparison of chemoattractants expression during MB49 bladder tumors grow highlighted CCL8 and CCL12 (CCR2-ligands), CCL9 and CCL6 (CCR-1-ligands), CXCL2 and CXCL5 (CXCR2-ligands), CXCL12 (CXCR4-ligand) and antagonist of C5/C5a as potential targets to decrease myeloid suppressive cells. Data obtained with a single CCR2 inhibitor however showed that the complex chemokine crosstalk would require targeting multiple chemokines for anti-tumor efficacy.
BackgroundMicrobial immunotherapy, in the form of intravesical Bacillus Calmette-Guérin (BCG), is the standard-of-care for non-muscle invasive bladder cancer (NMIBC). BCG therapy is associated with significant side-effects, high disease recurrence and progression rates, and product supply shortages, leaving a significant unmet medical need for bladder cancer patients.1 2 We sought to establish the preclinical safety and efficacy of live-attenuated Salmonella enterica Typhi strain ZH9 (ΔaroC, ΔssaV) as a novel microbial immunotherapy.MethodsTherapeutic efficacy of intravesical ZH9 was established in the murine orthotopic, syngeneic MB49 bladder tumor model. Tumor-bearing animals were treated with a single intravesical dose of ZH9 or OncoTice BCG and long-term survival comparisons were evaluated by log-rank (Mantel-Cox) test. ZH9 interaction with urothelial cancer cells was investigated using in vitro invasion assays and flow cytometry staining for intracellular Salmonella common antigen (CSA-1) and propidium iodide for cell death. Local immune responses were analyzed by flow cytometry staining of disaggregated mouse bladders.ResultsMice treated with a single intravesical dose of ZH9 2 days after MB49 tumor inoculation demonstrated significant survival benefit compared to vehicle treated (median survival 49.5 vs. 31 days, p=0.003) and BCG treated animals (median survival 49.5 vs. 27.5 days, p<0.001). A second, stringent model setup with intravesical treatment 4 days after tumor inoculation showed significant efficacy of ZH9 versus vehicle and BCG (median survival 30 vs. 20.5 (p=0.003) and 23.5 (p=0.025) days, respectively). Surviving ZH9 treated animals demonstrated 100% protection from tumor take following repeated intravesical challenge with MB49 tumor cells, suggesting lasting anti-tumor immunity resulting from ZH9 treatment. In vitro, intracellular flow cytometry in human (UMUC3, T24, RT4, 5637) and mouse (MB49) urothelial cancer cell lines showed that ZH9 invaded and induced cell death in all cell lines. In vivo, a single treatment with intravesical ZH9 resulted in strong cellular immune responses characterized by recruitment of monocytes, NK cells, CD4+ and CD8+ T cells, and dendritic cells with an activated, cross-presenting (Ly6C+, CD103+) phenotype. Intravesical ZH9 resulted in a greater magnitude and duration of immune cell recruitment in the urothelium compared to a single equivalent dose of intravesical BCG.ConclusionsLive-attenuated Salmonella strain ZH9 demonstrated a significant survival benefit over the standard-of-care OncoTice BCG in an orthotopic bladder cancer model. ZH9 demonstrated direct tumour cell killing in vitro and induction of robust cellular immune responses in vivo. Preclinical studies indicate significant therapeutic potential of intravesical ZH9 as a novel microbial immunotherapy in bladder cancer.ReferencesLiu Y, Lu J, Huang Y, Ma L. Clinical spectrum of complications induced by intravesical immunotherapy of Bacillus Calmette-Guérin for bladder cancer. J Oncol 2019 March 10;2019:6230409.vanRhijn BW, Burger M, Lotan Y, et al. Recurrence and progression of disease in non-muscle-invasive bladder cancer: from epidemiology to treatment strategy. Eur Urol 2009 September;56(3):430–42.Ethics ApprovalThe studies were conducted under approval from Pennsylvania State College of Medicine Institutional Animal Care and Use Committee approval number 47682, or with approval of the Cantonal Veterinary Office of Canton de Vaud, Switzerland.
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