EFFECTS OF LACTOBACILLUS ON NORMAL AND TUMOUR BEARING MICE SEOW SHIH WEE B.Sc (HON.) NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF SURGERY NATIONAL UNIVERSITY OF SINGAPORE Acknowledgements I would like to extend my heartfelt gratitude to my supervisors, Dr Ratha Mahendran, Prof Bay Boon Huat and A/P Lee Yuan Kun for their direction and invaluable advice throughout my candidature and during the process of producing this dissertation. Survival throughout the entire duration of the candidature could not have been possible without the help of all members of the lab, both past and present, some of whom became firm friends of mine. Special thanks to Juwita, Rachel, Shirong and Mathu for the laughter and support which never failed to come when most needed. Many thanks also to Mrs Ng Geok Lan, Poon Zhung Wei and Ms Pan Feng from the Immunohistochemistry laboratory (Anatomy), Ms Chan Yee Gek (Electron Microscopy Unit), and Mr Low Chin Seng (Microbiology) for their assistance and for imparting their lab skills to me. Finally, this dissertation is dedicated in its entity to my husband and parents from whom I draw strength for sustenance and determination. THANKS! i Acknowledgments i Table of contents ii List of Abbreviations viii List of Figures xi List of Tables xiii List of publications and conference papers xiv Summary xvi Introduction 1.1. Bladder cancer – an overview 1.2. Bladder cancer therapy 1.2.1. Surgery 1.2.2. Intravesical chemotherapeutic agents 1.2.3. Bacillus Calmette-Guérin (BCG) Immunotherapy of Bladder Cancer 1.2.4. ImmuCyst® [Bacillus Calmette-Guérin (BCG), substrain Connaught] 1.3. Mechanisms of BCG action 1.3.1. Fibronectin-mediated 1.3.2. Recruitment of immune cells 1.3.3. Pro-inflammatory cytokines 1.3.4. BCG viability influences treatment efficacy 1.4. Side-effects associated with BCG immunotherapy 11 1.4.1. Attempts to alleviate BCG side-effects 11 1.5. Probiotics and Lactobacillus species 12 1.5.1. Beneficial health properties of Lactobacillus species 13 1.5.1.1. Women’s reproductive and bladder health 13 1.5.1.2. Alleviating allergies 13 1.5.1.3. Boosts overall immunity 14 1.5.1.4. Ensuring good gastrointestinal health and prevention of 15 gastrointestinal infections 1.6. The genus Lactobacillus and cancer 16 1.6.1. Postulated anti-cancer mechanisms of Lactobacillus species 17 1.6.1.1. Alteration to gut microflora 17 ii 1.6.1.2. Alteration to metabolic activities of gut microflora 17 1.6.1.3. Adsorbing and facilitating excretion of carcinogens 17 1.6.2. Lactobacillus species and bladder cancer 20 1.7. Unanswered questions and inconsistencies in current reports 21 1.8. Lactobacillus rhamnosus strain GG (LGG) 22 1.9. Animal models of bladder cancer 22 1.10. Types of cell death in cancer treatment 28 1.10.1. Apoptosis 28 1.10.1.1. Nonsteroidal anti-inflammatory drugs (NSAID) activated gene 29 1.10.2. Necrosis 29 1.10.3 Autophagy 30 1.11. Scope of study 32 2. Materials and Methods 33 2.1. Bacteria culture 34 2.1.1. Lactobacillus rhamnosus strain GG (LGG) 34 2.1.2. Heat-killed LGG 34 2.1.3. Lyophilised LGG (lyo LGG) 34 2.1.4. LGG-green fluorescent protein (LGG-GFP) 35 2.1.5. Live Bacillus Calmette Guerin (BCG) 35 2.2. Tumour cell lines 36 2.3. Animals 36 2.3.1. Orthotopic procedures 36 2.3.2. Assessing the safe use of LGG in mice 37 2.3.2.1. LGG localisation and translocation 37 2.3.2.2. Immune cell population changes after bacteria instillations in healthy 37 mice 2.3.2.3. Expression of inflammatory cytokines and receptors after LGG 40 instillation in healthy mice 2.3.2.4. Reverse transcriptase polymerase chain reaction (RT-PCR) 44 2.3.2.5. Optimising LGG instillation schedule 48 2.3.3. Orthotopic tumour model 49 iii 2.3.3.1. Tumour implantation 49 2.3.3.2. Free Prostate Specific Antigen (f-PSA) Chemiluminescence 49 Immunoassay Kit 2.3.3.3 Monitoring tumour implantation efficiency and disease progression 50 2.3.4. Treatment of bladder cancer 51 2.3.4.1. Intravesical therapy with bacteria 51 2.3.5. Metastasis confirmation 52 2.3.6. Analysis of TNFα, TGFβ and IL10 expression in local lymph nodes 53 2.3.7. Urinary cytokines 54 2.3.8. Bladder protein isolation 55 2.3.8.1. Analysis of cytokines in bladder post-microbe instillations 55 2.3.8.2. Confirmation of cytokine protein array data with ELISA 56 2.3.9. Immunohistochemistry 57 2.4. Re-selection of tumour cell line 59 2.5. Co-culture of LGG with mammalian cells 59 2.5.1. In vitro stimulation of splenocytes with live or lyo LGG 60 2.5.2. Effects of LGG on MB49 cell proliferation 60 2.5.3. Cell cycle analysis 60 2.5.4. 62 2.5.5. Nonsteroidal anti-inflammatory drugs (NSAID) activated gene (NAG-1) real-time PCR Caspase-3 activity assay 62 2.5.6. Electron microscopy 64 2.6. Statistical analysis 65 3. Results 66 3.1. Assessing the persistence and immunomodulatory effects of LGG 67 3.1.1. Live LGG up regulates TNFα expression in splenocytes 67 3.1.2. Persistence of LGG in the bladder and other tissues after one and six 68 instillations 3.1.3. Comparing the ability of LGG and BCG to stimulate cytokine and 70 chemokine gene expression in the bladder 3.1.3.1. General observations 70 iv 3.1.3.2. Analysis of mouse inflammatory cytokines and receptors with 71 microarray 3.1.3.3. Analysis of mouse inflammatory cytokines, chemokines and 74 receptors with RT-PCR 3.1.3.4. Immune cell recruitment to the local lymph nodes and bladder 78 3.2. Modulating LGG’s immunogenicity through lyophilisation 80 3.2.1. Lyophilised LGG remains viable after lyophilisation 80 3.2.2. Live and lyo LGG stimulates cytokine mRNA and protein 80 expressions 3.2.3. Lyo LGG instillations did not result in host morbidity or mortality 84 3.2.4. Effect of one or two Lyo GG instillations a week on gene expression 84 in the bladder 3.2.5. Lyo LGG instillations attract activated and mature dendritic cells to 87 the bladder 3.2.6. Lyo LGG instillations changed the immune cell populations of the 88 local lymph nodes 3.3. Assessing and evaluating the anti-tumour efficacy of LGG in vivo 90 3.3.1. Monitoring orthotopic tumour implantation and disease progression 91 3.3.2. General observations 92 3.3.3. LGG instillations conferred survival advantage 92 3.3.4. LGG therapy conferred protective effect over PBS 93 3.3.5. Elucidating LGG’s anti-tumour mechanisms 93 3.3.5.1. Analysis of bladder proteins post-LGG therapy 93 3.3.5.2. Bladder protein ELISA 98 3.3.5.3. Profiling systemic and local immune response post-LGG therapy 101 3.3.5.4. Immune cell population in the local lymph nodes after LGG therapy 102 3.3.6. Histopathological and immunohistochemical analysis of Control PBS 103 and lyo LGG-instilled bladders 3.3.6.1. Histopathology and immunohistochemistry 104 3.3.6.2. Lyo LGG mobilised large numbers of neutrohpils and macrophages 105 into the tumour v 3.4. Comparing the efficacy of lyophilised LGG as an 109 immunotherapeutic with BCG 3.4.1. Re-selection of tumour cell line 109 3.4.2. A comparison of treatment efficacy between BCG Immucyst and lyo 111 LGG 3.4.2.1. General observations of Immucyst-treated mice 111 3.4.2.2. Lyo LGG is as efficacious as Immucyst in treating bladder cancer 111 3.4.2.3. Lyo LGG and Immucyst and tumour metastasis 113 3.4.3. Lyo LGG and BCG instillations did not alter urine TNFα and IL10 114 levels 3.4.4. Lyo LGG increased TNFα mRNA expression in local lymph nodes 116 3.5. Analysis of direct cytotoxic effects of LGG 119 3.5.1. Live LGG but not heat-killed LGG induces cytotoxicity 119 3.5.2. Live LGG inhibits murine and human bladder cancer cell 120 proliferation LGG induces sub-G1 population in the absence of direct contact with 3.5.3. 121 cancer cells 3.5.4. Lyo LGG is as efficacious as live LGG in inducing a large sub-G1 122 population 3.5.5. LGG increases NAG-1 mRNA expression in MGH cells 122 3.5.6. LGG did not induce caspase-3 activity 125 3.5.7. Live and lyo LGG induces cell death in MGH cells 125 4. Discussion 129 4.1. LGG as a non-pathogenic intravesical immunotherapeutic 131 4.2. Lyophilised LGG - a better immunostimulant than whole live 136 LGG 4.3. cRNA array versus semi-quantitative polymerase chain reaction 140 4.4. Treating bladder cancer with Lactabacillus rhamnosus strain GG 141 LGG’s indirect killing mechanisms 142 4.5.1. Role of proteins 142 4.5.2. Role of immune cells 145 4.5. ` vi 4.6. Effects of LGG in a healthy versus diseased bladder 149 4.7. LGG’s direct killing mechanisms 150 4.8. Lactic acid is not the cytotoxic metabolite 152 4.9. Translating in vitro evidence to in vivo tumour models 153 4.10. Conclusion 154 4.11. Future directions 155 References 158 vii List of Abbreviations (in alphabetical order) APC Allophycocyanin BCG Bacillus Calmette-Guerin BSA Bovine serum albumin Beta-defensin β-Def-1 CARE Centre for Animal Resources Ccl Chemokine (C-C motif) ligand CD Cluster of Differentiation protein CD3-FTIC Monoclonal Antibody to CD3, Fluorescein isothiocyanate (FITC) conjugated CD4-PE Monoclonal Antibody to CD4, Phycoerythrin (PE) conjugated cDNA Complementary deoxyribonucleic acid CFU Colony Forming Units CIS Bladder carcinoma in situ Cxcl Chemokine (C-X-C motif) ligand DAB 3,3'-diaminobenzidine DEPC Diethyl pyrocarbonate DR Death Receptor ELISA Enzyme-linked immunosorbent assay FANFT N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide FBS Fetal bovine serum Fc epsilon receptor Fcεr1g Fc gamma receptor Fcγr1 Foxp3 Forkhead box P3 GA Glutaraldehyde GAG glycoaminoglycan GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase GMCSF Granulocyte-Macrophage Colony-Stimulating Factor H&E Hematoxylin & Eosin Staining IACUC Institutional Animal Care and Use Committee ICAM-1 Intracellular adhesion molecule Interferon gamma IFNγ Ig Immunoglobulin IHC Immunohistochemistry ILInterleukinIL3Rb Interleukin receptor beta LPS Lipopolysaccharide iNOS Inductible nitric oxide synthase IP-10 Interferon-inducible protein 10 LAB Lactic acid bacteria LAKs Lymphokine-Activated Killer cells LcS Lactobacillus casei strain Shirota LGG Lactobacillus rhamnosus strain GG LGG-GFP LGG-green fluorescent protein LIX Chemokine (C-X-C motif) Ligand Lyo LGG Lyophilised LGG viii List of Abbreviations (continued) MAKs Macrophage-Activated Killer cells MBT-2 Mouse bladder tumour MHC Major Histocompatibility Complex MIP2 Macrophage inflammatory protein MRS de Man, Rogosa, Sharpe MMC Mitomycin C NAG-1 Nonsteroidal anti-inflammatory drugs (NSAID) activated gene NK Natural Killer cells NUS National University of Singapore OPN Osteopontin O+I Oral & intravesical therapy group PARP Poly (ADP-ribose) Polymerase PBS Phosphate buffered saline PF4 Platelet factor PG RPMI complete media with Penicillin G (5000units/ml) PLL Poly-L-lysine PMN Polymorphonuclear cells Pro-MMP9 Matrix metalloproteinase 9, pro-form PS RPMI complete media with Penicillin G (5000units/ml) and Streptomycin (5mg/ml) PSA Prostate specific antigen PtdSer Phosphotidylserine RANTES Regulated upon Activation, Normal T-cell Expressed, and Secreted, also known as Ccl5 RBC Red blood cell RNA Ribonucleic acids RT Room temperature RT-PCR Reverse transcriptase polymerase chain reaction RAC1 Ras-related C3 botulinum toxin substrate SCID Severe Combined Immunodeficiency Scye1 Small inducible cytokine subfamily E, member SD Standard deviation sTNF RI Soluble tumour necrosis factor receptor inhibitor I TCC Transitional Cell Carcinoma Tumour Growth Factor, beta TGF-β Th1 Helper T cell responses Thiotepa N,N'N'-triethylenethiophosphoramide Thymus Ck1 Thymus chemokine TIFF Tagged Image File Format TLR Toll-like receptor Tumour Necrosis Factor, alpha TNFα TUR Transurethral resection TMB 3, 3’, 5, 5’-tetramethylbenzidine TBS Tris-buffered saline ix 99 Oliveira1 PA, Colaço1 A, De la Cruz P LF, Lopes C. 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Cancer Res. 37: 546 - 50. 1977. 190 [...]... to Control tumour- bearing mice Array 4.1 - Proteins found to be expressed with a 2-fold difference with respect to Control tumour- bearing mice Comparison of bladder proteins from mice after 6 weeks of therapy Immune cell populations in spleen after various LGG therapies Comparisons in Mac-3+, CD19+ and Ly6G+ cell population between cured and tumour- bearing mice PSA concentration of the five colonies... stimulated with live and lyo LGG 2 instillations/week induced more cytokine/ chemokine mRNA than 1 instillation/week H&E staining of a section of a tumour- bearing bladder Survival rates of tumour- bearing mice after various LGG treatments X-ray images of Arrays 3.1 and 4.1 Representative H& E tissue sections of tumour- bearing bladders Representative images of lyo LGG and control bladder sections stained with... mAb Photomicrographs of bladder sections stained with antimouse Mac-3 mAb (macrophage) Monitoring growth of subcutaneous tumour Kaplan Meier analysis of BGC and lyo LGG therapy on the survival of tumour- bearing mice Urinary TNFα and IL10 1 week after lyo LGG instillations Microbe instillations elevated TNFα, TGFβ and IL10 transcript expression in local lymph nodes Total number of MGH cells remaining... properties of Lactobacillus species 1.5.1.1 Women’s reproductive and bladder health Lactobacillus strains are able to colonise the vagina following vaginal suppository insertion and reduce the risk of urinary tract infection, yeast vaginitis and bacterial vaginosis The rate of urinary tract infection in 25 women was compared before and after regular lactobacillus usage Prevention of urinary tract infection... live and lyo LGG therapy was found to confer a 30% survival advantage over controls (p < 0.05) xvi Oral feeding of live LGG in addition to lyo LGG instillations, did not augment lyo LGG’s anti -tumour efficiency The mice were typically cured of bladder tumour between the 2nd and 3rd instillations Lyo LGG instillations led to massive numbers of neutrophils and some macrophages infiltrating the tumour. .. European National Societies of Immunology 16th European Congress of Immunology 6 – 9 Sep 2006, Paris, France 2 A comparison of immune cells mobilisation after intravesical instillations of Mycobacterium bovis, Bacillus Calmette Guerin (BCG) and Lactobacillus rhamnosus strain GG (LGG) in mice (PD-3832) 1st Joint Meeting of European National Societies of Immunology 16th European Congress of Immunology 6 – 9... comparison of the immunomodulatory effects of Lactobacillus rhamnosus strain GG and Mycobacterium bovis, Bacillus Calmette-Guerin following instillations in healthy mice Federation of Clinical Immunology Societies – FOCIS 2006 01 – 05 Jun 2006, San Francisco xiv 4 Immunomodulatory effects of Lactobacillus rhamnosus strain GG in Healthy Mice (P109) Combined Scientific Meeting; Singhealth, National Healthcare... model of bladder cancer (Manuscript in preparation) 3 Seow SW, Bay BH, Lee YK and Mahendran R Understanding the anti -tumour mechanisms of Lactobacillus rhamnosus GG – in vitro (Manuscript in preparation) Conference Papers Poster presentation 1 An in vivo study of the immunotherapeutic potential of Lactobacillus rhamnosus GG in healthy murine bladders (PD- 2774) 1st Joint Meeting of European National... like dendritic cells and macrophages present BCG epitopes to T-helper cells culminating in T-cell activation [25] and cytokine production Polymorphonuclear (PMN) cells are early innate immune cells and the predominant subpopulation of leukocytes in the urine after BCG instillation [26] They migrate to the tumour site site after BCG instillation and mediate the recruitment of monocytes and CD4+ T-cells... experiments on normal healthy mice Treatment schedule for the orthotopic bladder tumour model Histogram of DNA content of healthy cells Live LGG induces TNFα expression Bacteria instillations led to enlarged local lymph nodes X-ray images of cRNA array Gene expression changes induced by the microbes Effect of live and lyo LGG on splenocyte TNFα and IL12p40 mRNA expression Cytokine production by splenocytes stimulated . cured and tumour- bearing mice. 103 Table 3.16 PSA concentration of the five colonies selected for subcutaneous tumour implantation. 110 Table 3.17 Odds ratio (OR) of mice bearing bladder tumour. EFFECTS OF LACTOBACILLUS ON NORMAL AND TUMOUR BEARING MICE SEOW SHIH WEE B.Sc (HON.) NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF SURGERY. 3.14 Monitoring growth of subcutaneous tumour. 110 Figure 3.15 Kaplan Meier analysis of BGC and lyo LGG therapy on the survival of tumour- bearing mice. 112 Figure 3.16 Urinary TNF α and IL10