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No difference was observed on the volume of inflammatory exudate, the amount of protein in the exudate, and connective tissue proliferation. The authors concluded that fish oil-derived fatty acids can probably modulate the chronic inflammation phase and cellular immune response, by inhibition of leukotriene B4 and prostaglandin E2 synthesis [ 42 ]. The results showed that anti-DLAA improves Ga uptake by tumor cells and that negative and neutral charge liposomes increase the accumulation of GA to tumor tissue as compared to positively charged liposomes.

Liposomes with Ga but without anti-DLAA had minimal accumulation in tumor tissue and maximal accumulation in the liver and spleen [ 43 ]. The authors observed loss of peptidoglycan and collagen, and formation of a granuloma, and tested different types of anti-inflammatory and antirheumatic drugs. They concluded that the air pouch model is better than other animal models to predict therapeutic efficacy in men [ 44 ]. Briefly, an air pouch model with carrageenan was induced in rats, and microspheres were administrated either orally or intravenously. The authors concluded that microspheres could be used as a vehicle for inflammation-targeted treatment [ 45 ].

They concluded that the vascular endothelial growth factor may be an important regulator of angiogenesis during the inflammation process [ 46 ]. Briefly, hairless mice were fed with celecoxib or indomethacin, and air pouches were exposed to ultraviolet light. The authors stated that the air pouch method is a highly sensitive method to test the response of inflammatory cells to biomaterials [ 49 ].

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Gene transfection was corroborated by PCR analysis. And, decrease of inflammation was observed in transfected mice by histological analysis. The authors concluded that the air pouch model is useful to evaluate gene therapy [ 50 ]. The results suggest that LT is probably more effective than not encapsulated anatase particles of titanium dioxide for the treatment of bladder cancer [ 52 ]. It was concluded that interleukin cannot start an inflammatory response, but it is able to increase inflammation in its early stages [ 53 ]. This group determined the immunoregulatory properties of Lactobacillus paracasei subsp.

This group evaluated biocompatibility, toxicity, and inflammatory and adaptive immunological response to nanoparticles designed for nanomedicine. They reported that this technique yields reproducible data, with three mice per test [ 55 ]. Honey was injected into the pouch at the same time as carrageenan and then for 2 consecutive days. The evaluation parameters included hemoglobin concentration, vascular endothelial growth factors and prostaglandin E2 levels, and granulomatous tissue weight. The results showed a decrease of angiogenesis, and it was concluded that honey might be useful in the treatment of granulomatous inflammatory conditions [ 56 ].

Twenty hours later cell infiltrates were harvested from the pouches and stimulated with phorbol myristate acetate PMA plus ionomycin and brefeldin A, and intracellular levels of interleukin were determined by flow cytometry analysis. We induced the air pouch model, inoculated LY-R cancer cells, and determined if our compound interfered with tumor implantation. We concluded that our bio-compound has antitumor properties [ 59 ].

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In general, a common process to induce air pouches is performed as follows: Sterile air is obtained in a laminar flow station by filtration through a Millipore filter 0. On day 8, the pouches of the experimental groups are filled with necessary doses of the compound to test, according to our requirements. Step-by-step sterile air filtration in laminar flow hood.

Air pouch formation. The rodent back is shaved A ; the animal is held, and air is injected subcutaneously into the back B ; and air pouch inflation is observed C and D. The air pouch completely formed E. Tumor cell inoculation into the air pouch. The skin from the air pouch B and tumor B were detached the arrow points to the tumor.

Tumor sections were stained with hematoxylin and eosin C. Cell number varies depending on the cell line.

Mouse Models of Cancer

Treatment administration scheme depends on the tested drug. We recommend adding a group treated with placebo injectable solution and with the reported treatment e. We recommend adding a group treated with placebo injectable solution and with the reported treatment group e. Note: For each experiment design, it is important to take into consideration variables that could alter the results, such as animal age and sex, and treatment administration.

The appropriate age range of the rodents depends on the experiment and relevant age for human disease. Animal sex: This can also affect the therapy outcome. This is particularly true for targeted therapies, and it has become a requirement for any applications for the National Institutes of Health NIH to report the male and female ratio in preclinical studies [ 62 ].

Genes encoded by the Y chromosome include inflammatory pathway genes, while X chromosome encodes genes for Toll-like receptors, cytokine receptors, and transcriptional and translational regulator genes. Although X chromosome is present in both sexes, female X polymorphism allows for mosaicism and randomly silenced alleles. It has been observed that male sex leads to data alteration in pharmacology and neuroscience, and female sex affects immunology results [ 62 , 63 ].

Therapy administration: Carcinogenesis is described as a three-stage process, initiation, promotion, and progression. Compounds that prevent or delay any stage of carcinogenesis are called chemopreventive agents. Chemoprevention is defined as the use of compounds to reduce the risk or delay the development of cancer or to avoid its recurrence. Bio-compounds such as food-derived polyphenols, usually inhibit initiation and promotion, of induced cancer, and therefore are considered chemopreventive agents [ 64 ]. When testing antitumor activity of natural or synthetic compounds, highly variable results can be obtained based on the type of tumor, the administration dose and frequency, and the evaluation parameters: tumor incidence, growth, or metastasis.

Therefore, the purpose and parameters of the preclinical testing must be clearly defined [ 65 ]. Angiogenesis: Angiogenesis is a physiological process that refers to new blood vessel formation. In the tumor microenvironment, angiogenesis is over-induced to sustain tumor growth and metastasis. The vascular endothelial factor is one of the parameters used to evaluate angiogenesis. This factor induces angiogenesis, and it is increased in tumor and tumor-adjacent stroma tissue and correlates with tumor aggressiveness and with the patient prognosis [ 66 ].

Another parameter for angiogenesis evaluation is the tissue hemoglobin content [ 56 , 57 ]. Angiogenesis can be measured in the tumor and adjacent tissue skin from the air pouch , with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry, ELISA, and RT-PCR.

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  • Cancer-associated fibroblasts CAFs : Fibroblasts are the most abundant cells of the connective tissue; they produce collagen for the extracellular matrix and are involved in wound healing. In the tumor microenvironment TME , cancer cells and other stroma cells induce fibroblasts to produce tumor-promoting substances such as epidermal and hepatocyte growth factors; chemokines CXCL12, CXCL14, and CCL5 [that attract immature and suppressor immune cells]; vascular endothelial growth factor; and interleukin-6, among others. This type of fibroblasts is termed activated or cancer-associated fibroblasts CAFs , and they correlate with tumor growth, progression, metastasis, and chemoresistance [ 67 ].

    Indoleamine 2,3-dioxygenase 1 IDO1 : It is a cytosolic enzyme that catabolizes tryptophan into kynurenine, a metabolite with immunosuppressive properties. Increased levels of IDO1 correlate with the decrease of natural killers and specific effector T cells and increase of regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells.

    IDO1 also correlates with tumor progression and multidrug resistance. It is therefore considered a tumor progression biomarker and a promising therapeutic target [ 68 ]. Lipid rafts: Cholesterol and sphingolipids form specific domains termed lipid rafts that regulate receptor-ligand interactions.

    In cancer cells, signaling protein and pro-oncogenic receptor activation correlates with their location inside the lipid rafts; disruption of lipid rafts induces apoptosis in cancer cell lines. Lipid rafts are characterized by the presence of glycosylphosphatidylinositol GPI -anchored proteins [ 71 ]. Lipid rafts can be measured in the tumor and adjacent tissue skin from the air pouch , with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry, and ELISA.

    Liver toxicity: Synthetic and biological compounds are often metabolized and excreted by the liver. Certain drugs can be metabolized to reactive compounds that bind to intracellular proteins inducing oxidative stress and cell death. Liver toxicity must be evaluated in early phase or preclinical studies of any drug and can be assessed by transcriptomics, cellular respiration, ATP adenosine triphosphate , ROS reactive oxygen species , covalent binding, apoptosis or necrosis, and bile salt export pump inhibition tests [ 73 ].

    Liver toxicity can be evaluated in the liver tissue by immunohistochemistry or Western blot or in peripheral blood by flow cytometry or ELISA. Furthermore, blood tests can be included glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase. MSCs have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes and are recruited to injured tissues for healing.

    Methods and Protocols

    MSCs can be evaluated in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy, Western blot, and immunohistochemistry. Tumor-associated macrophages: Macrophages are specialized phagocytic cells of the immune system. In response to various stimuli, macrophages shift their phenotype to M1 or M2. M1 macrophages are associated to an inflammatory response with antitumor properties; on the other hand, M2 macrophages promote tumor growth and have anti-inflammatory properties.

    TAMs can be evaluated in the tumor tissue skin from the air pouch , with the following techniques: flow cytometry, fluorescence microscopy, Western blot, and immunohistochemistry. Tumor growth rate: Tumor size is defined by the Response Evaluation Criteria in Solid Tumors RECIST as the sum of the longest diameters of the tumor mass; to report the tumor growth rate, tumor size can be measured during relevant therapy time points, for example, before treatment, after the first treatment cycle, after the last cycle of treatment, and after discontinuation of the treatment [ 76 ].

    Also, the Ki67 levels, a protein in all phases of the cell cycle G1, S, G2, and mitosis , except for the resting phase G0 , often correlate with tumor growth and progression [ 77 ]. Tumor growth can be measured with a caliper volume , or weighted after removal, or determination of Ki67 levels in the tumor tissue by flow cytometry, fluorescence microscopy, Western blot, and immunohistochemistry.

    To be clinically successful, an anticancer drug must have effect over cancer cells in the tumor microenvironment context; however, in vitro models do not include all of its components. Therefore, the improvement of cancer models is considered a priority for the current drug development [ 55 ]. In the current chapter, we propose a modified air pouch model as an alternative or complement to in vitro studies. As previously described, the air pouch model has been extensively used to evaluate inflammation process, anti-inflammatory compounds, immune response, biomaterial compatibility, and of course cancer development and treatment.

    The air pouch model allows the administration of higher volumes of chemotherapy alone or combined with other treatment modalities, including targeted therapy, immunotherapy, and biological compounds, to determine single or cumulative antitumor effects, simulating the true clinical condition and treatment.

    Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. What has been missing in the TNBC mouse model is a sequential progression of the disease in an essential native microenvironment. We established that the TNBC-MIND model in syngeneic mice could epitomize all breast cancer progression stages and metastasis into the lungs via lymphatic or hematogenous dissemination within four weeks.


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    This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: All relevant data are within the paper and its Supporting Information files.

    Competing interests: The authors have declared that no competing interests exist.