Animal BT Studying Notes PDF

Describe the pathogenesis and process of cancer. Tumorgenesis - complex process involving the accumulation of multiple genetic aberrations that transform normal cells, allowing for their abnormal growth, proliferation, & metastasis. 1) Initiation 2) Promotion 3) Progression 4)...

Describe the pathogenesis and process of cancer. Tumorgenesis - complex process involving the accumulation of multiple genetic aberrations that transform normal cells, allowing for their abnormal growth, proliferation, & metastasis. 1) Initiation 2) Promotion 3) Progression 4) Metastasis Many animals are considered to be used as cancer disease models. Mouse in particular is the most common among them. What are the advantages and limitations of mouse models in cancer research? Advantages Limitations Small size; easy to handle Different in terms of size, life span, organ morphology & physiology; differs from PK and PD data Cheaper than others animal models Differences in telomerase enzymes, which is largely inactive in adult human cells High tumor incidence & rapid tumor growth Tend to develop few metastases Many mice can be treated at the same time to Differences in metabolic rate & pathways - observe dose responses result in different drug response Genetically, best characterized of all Limited number of initiating genetic mammals used in cancer research alterations - mouse tumors are more homogeneous - obstacles to model the heterogeneity of human cancers What kind of genetic background mice are typically used in cancer research? Inbred mice - all the mice have identical genetic background & are expected to generate similar responses to treatments. BALB/c mice.  What are the different types of experimental tumor mouse models used for cancer immunology research? Characteristics Transplantable tumor Genetically engineered Humanized Models models tumor models (GEMMs) Definition Tumour is transplanted Tumours arise Immunodeficient animals into host animals, often spontaneously in animals engrafted with human derived from cell lines due to the genetic cells/tissues for cancer or patient tissues modifications study Tumor Origin Derived from Arises endogenously from Derived from human cancer established cancer cell engineered mutations in cells or tissues transplanted lines or patient-derived specific genes into humanized mice xenografts (PDX) Immune System May be intact or Fully functional From human stem cells or suppressed peripheral blood cells What are the different types of transplantable tumor models? Discuss their advantages & limitations. Characteristics Allograft (Syngenic) Model Xenograft (PDX) Model Definition Tumors or cancerous cells are Human tumor cells are itself of mouse origin, and are transplanted into a host, either transplanted into another host under the skin or into the mouse containing a specific organ type (orthotopic) in genetic trait which the tumor originated Tumor Source Mouse cancer cell lines or Human cancer cell lines or tissues patient-derived xenografts Host Species Immunocompetent mice Severely compromised immunodeficient (SCID) // immunocompromised mice Advantages - Results can be - Allows personalized analyzed in an cancer studies environment that - Mimics human tumor closely recapitulates genetics and biology the real scenario  where tumor grows in an immunocompetent environment Limitations - May not represent in - Higher cost and the whole complexity technical complexity of human tumors in - Requires clinical situations immunocompromised host, eliminating immune response studies Applications Preclinical and clinical Toxicity studies from development of anti-cancer targeted therapies, and therapeutics in many cases to predict biomarkers of target modulation What are the steps towards preparing transplantable tumor models? 1) Tumors are obtained and kept viable in the frozen state for prolonged periods 2) Immerse small pieces of tumor in media such as glycerol-glucose, in sealed sterile ampoules 3) The tumor-medium mixture should be slowly cooled to final storage temperature and rapidly thawed on removal, and after thawing, tumors should be immediately inoculated in mice 4) Depending on the number of cells injected, once the tumor develops to an appropriate size the response to therapeutic regimes can be studied in vivo. What are the different types of genetically engineered mouse models? Characteristics Retrovirus-Mediated Microinjection Targeted manipulation of mouse embryonic stem (ES) cells Definition Uses retrovirus to introduce Direct injection of Targets specific cells in mouse genetic material into host foreign DNA material ES cells, followed by cells, leading to random into a fertilized mouse selection and induction into integration zygote developing embryo Integration Site Random Integration Random Integration Precise, targeted integration at specific loci Advantages - Simple and quick to - Allows generation - High precision in  perform of transgenic targeting specific - Effective for creating animals in one genes random mutagenesis generation - Can create models - High versatility loss-of-function and for expressing conditional models transgenes - Preserves genetic background Limitations - Random integration - Random - Time-consuming & can disrupt essential integration can technically genes or regulatory result in variable challenging regions expression - Requires advanced molecular techniques What are the applications of GEMMs? 1) GMMs drive neoplastic transformation of normal cells to drive tumor growth, this gradual development & progression of cancer allows for the autochthonous development of a complex tumor microenvironment 2) The de novo tumour microenvironments that develop in the context of GEMMs contain native immunosuppressive stroma & vasculature, both critical variables that determine the magnitude and composition of immune cell infiltration What are the general limitations of GEMMs? 1) Penetrance of the tumour phenotype & latency of neoplastic development, which can vary dramatically depending on the mechanisms used to induce tumor development. 2) It is necessary to utilize non-invasive imaging modalities, such as ultrasound or magnetic resonance imagining, to monitor tumor development, standardize treatment scheduling, & monitor kinetics of antitumor immune responses. 3) Important to consider whether the murine immune target is cross-reactive with the corresponding human target. This includes antigens & surface markers that are present on human immune cells or tumors that are not present on murine cells. What is the gene construct that is used for gene targeting by homologous recombination? - Neomycin resistance gene as the positive selective marker - knockout gene function - TK gene as the negative selective marker - to initiate cell death for those with random integration of gene construct. Discuss with process of Targeted manipulation of mouse embryogenic stem cell with the neor and TK gene construct. Key points: 1) selection of transgenic cell (neor & TK gene) 2) Injected selected cell line into blastocysts and transfer to surrogate mouse 3) Obtain chimeric mouse and cross with wild type to obtain heterozygous genetically engineered mouse 4) Cross with each other to obtain homozygous genetically engineered mouse. What kind of gene construct is used for conditional gene modification? Cre-Lox technology: each line expressing Cre from a promoter that is either tissue specific, cell specific, developmentally specific or responsive to exogenous agent like tetracycline Discuss the process of the Cre-Lox Technology. Key points: 1) Two different lines, Cre-line and Floxed line 2) Floxed line, flanks the gene which we want to knockout 3) Floxed line crossed with Cre-line; then progeny which will target the specific tissue What kind of mice are used in humanized tumor models? Critical factor is the degree of immunodeficiency of the murine host: 1) T- & B- cell development and function 2) NK cells NSG mouse, a preclinical model with engineered combined immunodeficiency, has been among the most frequently used hosts for chimeric human-mouse immune reconstitution as well as other tissue chimeras. How do humanized tumor models work? Key points: 1) CD34+ hematopoietic stem cells from patients were injected to NSG mouse 2) Leading to the development of immune systems and human hematopoietic. 3) The immunodeficient mouse is then reconstituted with human DCs, T, B and NK cells. 4) Alongside the patient’s tumor 5) Making the mouse more humanized What are the applications of humanized tumor models? - Model the complexity involved in the natural tumor development - Genomic heterogeneity - Tumor architecture - Microenvironment factors - which is critical to develop an effective in vivo preclinical tumor model for therapeutic evaluation What are the limitations of humanized tumor models? 1. Requires high take rates for successful propagation of tumors across multiple mice within a reasonable timeframe 2. Repeated invasive sampling for hematopoietic cells would be necessary to develop individual humanized PDX mice 3. Patient survival times and resultant access to hematopoietic blood cells could be limited, thus lowering the pragmatic feasibility of this approach  Compare the advantages and limitations of the experimental tumor models used for cancer immunology research. Experimental model Strength Limitations Transplantable tumors models - Simply - Limited recapitulation of - Rapid of tumor formation & TME evaluation of therapeutic responses GEMMS - Tumours harbour genetic - Long time period for tumor lesions found in human development & evaluation cancers of therapeutic response - Improved modelling of tumor - Increase cost & complexity progression & TME of generating & maintaining - Tumour-induced tolerance & transgenic mice immunosuppression show similarities to human cancers Humanized Mice - Human immune populations - Logistically difficult to studied directly obtain matched donor and - Might able to capture tumor samples heterogeneity of patient - Incomplete reconstitution of responses human immune response - Expensive What are the current directions for transgenic mouse cancer models? 1) Personalizing humanized mice 2) Replicating specific human cancer mutations in mouse models 3) Improving delivery methods