**2. Model review**

followed by animal and finally human trials [1]. However, only about 5% of anticancer drugs finally get approved for clinical use due to lack of clinical efficacy or intolerable toxicity [1, 2]. In vitro approach contribution for biotechnological development is undeniable; however, cell lines are maintained in nonphysiological conditions that do not resemble body temperature, electrolyte concentration, extracellular matrix contact, cell density, and heterogeneity. Culture conditions also implicate sudden changes such as media exchange and nutrient depletion. Furthermore, rapid cell growth is desirable and induced, driving cell subpopulations not to differentiate. All of these factors alter cell signaling and favor specific subpopulations of cells that adapt to these artificial conditions and loose some of their original characteristics [3].

To optimize cancer drug screening, it is necessary to include the biological and genetic components that influence cancer treatment outcome. Therefore, to study the cancer cell in vivo, it is essential to understand how to prevent the establishment, growth, progression, and metastasis of cancers and how to modulate the tumor microenvironment (TME) for therapeutic gain [4]. Mouse models have been used to assess toxicology and efficacy of anticancer drugs, as well as for the study of tumor induction (establishment), progression, and metastasis; these traditional mouse models have been successfully used to identify cytotoxic drugs that are still the

In this chapter, we discuss about the benefits of a tumor model in which tumors can grow inside an air pouch created in the dorsal part of the mouse (**Figure 1**). The air pouch cancer model serves as the local microenvironment, which can be modulated to study establishment,

**Figure 1.** Mouse air pouch model for the study of cancer scheme. The general methodology to induce the air pouch model for the study of cancer and its advantages is depicted. Briefly, 5 milliliters of sterile air is injected into the shaved back of a mouse; after 3 days the pouch is reinflated with 5 milliliters of sterile air. After 3 days tumor cells are inoculated

progression, and metastasis as well as the efficacy of therapeutic agents.

main anticancer treatment in therapy today [4, 5].

172 Cell Culture

and allowed to grow prior to drug administration.

1953: The air pouch model was described by Selye for the study of the mechanism of action of hydrocortisone. Selye termed his model the granuloma pouch model and described it as a "procedure designed to permit the objective, quantitative analysis of factors regulating inflammation and wound-healing" [6].

1954: Kleinfeld and Habif evaluated the anti-inflammatory effect of trypsin and chymotrypsin inside a granuloma pouch. According to the authors, this method provides a standardized and reproducible inflammatory lesion. They evaluated the volume of the exudate and the weight of the pouches. The authors concluded that parenteral administration of trypsin and chymotrypsin does not affect the formation of granulation tissue in a chronic sterile inflammation environment [7].

1956: Hewitt observed that the number of viable cancer cells needed to induce tumors in half of an experimental group of adult mice was 1641 and in newborn mice was 10. He hypothesized that tumorigenesis could be affected by the dispersion of cancer cells and by the vascularity of the injection site. To test this hypothesis, he injected tumor cells into subcutaneous tissue and into an air pouch, and to evaluate the vascularity effect, he induced hyperemia in air pouches with a formic acid solution. Hewitt concluded that neither cancer cell dispersion nor vascularity affected the capacity of cancer cells to induce a tumor. He also suggested the use of the air pouch technique for further tumor transplantation studies [8].

1957: Selye wrote another article this time addressing the question: is inflammation good or bad for cancer development? He induced two air pouches on the back of the same animal, administered croton oil into one of the pouches to establish an inflammatory environment, inoculated cancer cells into both pouches, and evaluated which tumor developed faster. The results showed that inflammation can promote tumor development [9].

1957: A scientific report stated that the air pouch model presented several advantages over other methods for the study of inflammation. According to Robert and Nezamis, the granuloma pouch technique is simple and yields uniform results, and it is quantifiable because the degree of inflammation is reported in grams of the tissue, thickness of the pouch membrane, or volume of exudate. There is no systemic response, and furthermore they recommend this technique as a screening test for unknown compounds because minimal amounts of substance can be tested with high sensitivity in a short period of time [10].

1957: Based on the observation that two concomitant tumors in the same animal inhibited or promoted their development, Hans Richer inoculated nonviable sarcoma cells and viable Walker tumor cells into a mouse air pouch, to determine if the growth inhibition between tumors was due to nutrient competition. The air pouch model was used in this study because, according to the author, it is an ideal method to test a localized effect. He concluded that nonviable sarcoma cells delayed tumor growth when injected previously or concomitantly with the viable Walker tumor [11].

1958: It was known that cortisol retarded the growth of Walker tumor in rats; to determine if the effect was due to a local or systemic action of the hormone, Selye used his air pouch technique. According to him, the "granuloma pouch technique forces the malignant tissue to grow in the form of a thin lining on the wall of a regularly shaped ellipsoid cavity to which accurately measured amounts of hormones can be applied." Selye concluded that tumor growth inhibition was considerably more pronounced when cortisol was administered inside the air pouch containing the tumor as compared to administration subcutaneously in another location. In the same article, the author suggests that the hormones probably do not act directly upon cancer cells, but rather modify the stroma and the vascularization [12].

1968: Another adaptation of the air pouch model was employed by Toto et al. to determine if artificially produced cell culture media could induce cell proliferation in vivo. This group administered tritiated thymidine into the mouse to evaluate the proliferation rate. Their results suggest that T199 tissue culture medium supports and promotes cell growth in vivo [19].

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175

1969: The air pouch model was used to examine the role of polymorphonuclear leukocytes in tissue repair. Briefly, an air pouch was formed in the back of mice, and turpentine was injected to cause acute inflammation. Polymorphonuclear leukocytes were extracted from this air pouch, labeled with tritiated thymidine, and inoculated into the pouch of a recipient mouse. Leukocyte cytolysis was detected and the tritiated thymidine was incorporated by DNA synthetizing fibroblasts. The authors concluded that the neutrophil degradation prod-

1969: Willis used the air pouch model to obtain large volumes of exudate to extract and quantify prostaglandins for further assays. He suggested that pharmacological activity of inflam-

1970: Anti-inflammatory activity of topically applied hydrocortisone acetate, methylprednisolone acetate, betamethasone 17-valerate, triamcinolone 16,17-acetonide, betamethasone, and fluocinolone acetonide was tested in an air pouch model. It was concluded that the air pouch model served as an application area and was used for the evaluation of commercially

1970: An ascitic variant of an induced hepatoma cell line was established. The original tumor was inoculated inside an air pouch. Tumor cells grew attached to the pouch walls, and in addition some cells accumulated in the fluid inside the pouch. The fluid was extracted and injected intraperitoneally in female mice and maintained for more than six passes. This method pro-

1973: Sensitivity of the capillary endothelium to radiation was tested. Briefly, an air pouch was induced in rats, then the area was depleted of blood vessels by freezing, and vascular proliferation was later induced employing uric acid and lithium lactate. The area becomes revascularized within 12 days. It was observed that in irradiated preparations, revasculariza-

1974: On the 30th symposium of the Society for Developmental Biology, the air pouch model

1974: The air pouch model was used to study the binding of carcinogen 7,12-dimethylbenz[a] anthracene (DMBA) to the DNA of the mouse epidermis. The results showed that it is possible to study molecular events, such as the binding of DMBA to replicative or non-replicative DNA in vivo. It was concluded that DMBA binds preferentially to non-replicating DNA [26]. 1974: An air pouch in the rat was the technique used to demonstrate that tumors can induce

1974: Bladder fragments and air were subcutaneously injected in the backs of Wistar rats; within a month a cyst completely lined by urothelial cells was formed and persisted for at

duces high amount of tumor cells free of non-tumor cells and connective tissue [23].

was listed as one of the bioassays to evaluate the tumor angiogenesis factor [25].

ucts are used by fibroblast to proliferate and repair damaged tissue [20].

matory exudates was attributed to E-type prostaglandins [21].

available corticosteroids [22].

tion decreased in a dose-dependent manner [24].

endothelial mitosis from 3 to 5 mm distance [27].

1958: Another adaptation of the air pouch model was used by Ship et al. to determine whether administration of local cytotoxic agents could prevent tumor recurrence after surgical interventions. For a better recreation of a surgical wound environment, Ship removed the air out of the pouches after tumor inoculation. He termed his modified model "the air bubble technique" and demonstrated the effectiveness of formaldehyde against tumor cells implanted in vivo [13].

1958: The granuloma pouch was used to study the role of histamine as a mediator of acute inflammation. The air pouch model was employed because, according to previous reports, croton oil used to form the granuloma pouch disrupted mast cells, therefore releasing histamine. The histological changes associated with inflammation were not observed when histamine was depleted. The author concludes that histamine is closely related to early inflammatory reactions [14].

1961: Thoracic duct lymph cells from donor rats were injected into an air pouch of a receptor rat, for cytological and histological studies of the lymphocyte. The inoculation of thoracic lymph duct cells into the air pouch also allows simultaneous injection of antigen, fatty substances, and other materials to stimulate development of plasma cells and macrophages, among others, and either donor or pouch animal can be pretreated to alter the lymph cells or their tissue environment [15].

1967: Chang, Gibley, and Ichinoe used the air pouch to study the histological features of early passages of a chemically induced hepatocarcinoma. The authors stated that the air pouch method provides easy access to the tumor for faster processing of the tissue for histochemical, ultrastructural, or biochemical studies [16].

1967: The effect of the hormones progestogen, estrogen, and chorionic gonadotropin on the inflammatory response was studied. The granuloma pouch technique (air pouch with croton oil injection) in rats was used as the inflammation model. The thickness of the granuloma pouch was the parameter used to measure inflammation. It was concluded in this study that progestogen, estrogen, and chorionic gonadotropin act as antiphlogistic substances on acute inflammation [17].

1968: Orchidectomized adult male rats received a subcutaneous transplant of ovarian grafts. The experiment determined that daily injections of progesterone (0.1 milligrams) and continuous mild stress (air pouch with croton oil) or higher doses of progesterone (0.25 milligrams) induce luteinization [18].

1968: Another adaptation of the air pouch model was employed by Toto et al. to determine if artificially produced cell culture media could induce cell proliferation in vivo. This group administered tritiated thymidine into the mouse to evaluate the proliferation rate. Their results suggest that T199 tissue culture medium supports and promotes cell growth in vivo [19].

1958: It was known that cortisol retarded the growth of Walker tumor in rats; to determine if the effect was due to a local or systemic action of the hormone, Selye used his air pouch technique. According to him, the "granuloma pouch technique forces the malignant tissue to grow in the form of a thin lining on the wall of a regularly shaped ellipsoid cavity to which accurately measured amounts of hormones can be applied." Selye concluded that tumor growth inhibition was considerably more pronounced when cortisol was administered inside the air pouch containing the tumor as compared to administration subcutaneously in another location. In the same article, the author suggests that the hormones probably do not act directly upon cancer cells, but rather modify the stroma and the vascularization [12].

1958: Another adaptation of the air pouch model was used by Ship et al. to determine whether administration of local cytotoxic agents could prevent tumor recurrence after surgical interventions. For a better recreation of a surgical wound environment, Ship removed the air out of the pouches after tumor inoculation. He termed his modified model "the air bubble technique" and demonstrated the effectiveness of formaldehyde against tumor cells implanted

1958: The granuloma pouch was used to study the role of histamine as a mediator of acute inflammation. The air pouch model was employed because, according to previous reports, croton oil used to form the granuloma pouch disrupted mast cells, therefore releasing histamine. The histological changes associated with inflammation were not observed when histamine was depleted. The author concludes that histamine is closely related to early inflammatory

1961: Thoracic duct lymph cells from donor rats were injected into an air pouch of a receptor rat, for cytological and histological studies of the lymphocyte. The inoculation of thoracic lymph duct cells into the air pouch also allows simultaneous injection of antigen, fatty substances, and other materials to stimulate development of plasma cells and macrophages, among others, and either donor or pouch animal can be pretreated to alter the lymph cells or

1967: Chang, Gibley, and Ichinoe used the air pouch to study the histological features of early passages of a chemically induced hepatocarcinoma. The authors stated that the air pouch method provides easy access to the tumor for faster processing of the tissue for histochemical,

1967: The effect of the hormones progestogen, estrogen, and chorionic gonadotropin on the inflammatory response was studied. The granuloma pouch technique (air pouch with croton oil injection) in rats was used as the inflammation model. The thickness of the granuloma pouch was the parameter used to measure inflammation. It was concluded in this study that progestogen, estrogen, and chorionic gonadotropin act as antiphlogistic substances on acute

1968: Orchidectomized adult male rats received a subcutaneous transplant of ovarian grafts. The experiment determined that daily injections of progesterone (0.1 milligrams) and continuous mild stress (air pouch with croton oil) or higher doses of progesterone (0.25 milligrams)

in vivo [13].

174 Cell Culture

reactions [14].

their tissue environment [15].

inflammation [17].

induce luteinization [18].

ultrastructural, or biochemical studies [16].

1969: The air pouch model was used to examine the role of polymorphonuclear leukocytes in tissue repair. Briefly, an air pouch was formed in the back of mice, and turpentine was injected to cause acute inflammation. Polymorphonuclear leukocytes were extracted from this air pouch, labeled with tritiated thymidine, and inoculated into the pouch of a recipient mouse. Leukocyte cytolysis was detected and the tritiated thymidine was incorporated by DNA synthetizing fibroblasts. The authors concluded that the neutrophil degradation products are used by fibroblast to proliferate and repair damaged tissue [20].

1969: Willis used the air pouch model to obtain large volumes of exudate to extract and quantify prostaglandins for further assays. He suggested that pharmacological activity of inflammatory exudates was attributed to E-type prostaglandins [21].

1970: Anti-inflammatory activity of topically applied hydrocortisone acetate, methylprednisolone acetate, betamethasone 17-valerate, triamcinolone 16,17-acetonide, betamethasone, and fluocinolone acetonide was tested in an air pouch model. It was concluded that the air pouch model served as an application area and was used for the evaluation of commercially available corticosteroids [22].

1970: An ascitic variant of an induced hepatoma cell line was established. The original tumor was inoculated inside an air pouch. Tumor cells grew attached to the pouch walls, and in addition some cells accumulated in the fluid inside the pouch. The fluid was extracted and injected intraperitoneally in female mice and maintained for more than six passes. This method produces high amount of tumor cells free of non-tumor cells and connective tissue [23].

1973: Sensitivity of the capillary endothelium to radiation was tested. Briefly, an air pouch was induced in rats, then the area was depleted of blood vessels by freezing, and vascular proliferation was later induced employing uric acid and lithium lactate. The area becomes revascularized within 12 days. It was observed that in irradiated preparations, revascularization decreased in a dose-dependent manner [24].

1974: On the 30th symposium of the Society for Developmental Biology, the air pouch model was listed as one of the bioassays to evaluate the tumor angiogenesis factor [25].

1974: The air pouch model was used to study the binding of carcinogen 7,12-dimethylbenz[a] anthracene (DMBA) to the DNA of the mouse epidermis. The results showed that it is possible to study molecular events, such as the binding of DMBA to replicative or non-replicative DNA in vivo. It was concluded that DMBA binds preferentially to non-replicating DNA [26].

1974: An air pouch in the rat was the technique used to demonstrate that tumors can induce endothelial mitosis from 3 to 5 mm distance [27].

1974: Bladder fragments and air were subcutaneously injected in the backs of Wistar rats; within a month a cyst completely lined by urothelial cells was formed and persisted for at least 6 months. This heterotypic bladder in the rat was described by the authors as the first step to study the implantation, progression, and spread of bladder cancer [28].

24 hours later, the tissue was excised, and sister chromatid exchange frequencies were determined. It was concluded that mitomycin C and cyclophosphamide induce sister chromatid

Air Pouch Model: An Alternative Method for Cancer Drug Discovery

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177

1983: The carcinogenic potential of procarbazine was evaluated in an air pouch model in rats. It was concluded that high doses of procarbazine (300 mg/Kg) injected into the subcutaneous tissue do not induce tumors, unless an inflammatory environment had been previously

1984: An air pouch model was induced beneath the mammary fat pad of Wistar rats and injected with 20 milligrams of DMBA (7,12-dimethylbenz[a]anthracene). This technique allowed the induction of localized, transplantable, and estrogen-dependent adenocarcinomas in 67–80 days as compared to 150–180 days required by other techniques. The authors suggested that this model might be useful to study the biochemical mechanisms involved in

1986: Sedgwick and Lees compared three different acute inflammation models: a 6-day air pouch, polyester sponge, and pleurisy. Overall, they concluded that the air pouch model was

1987: Pretreatment of mice with methotrexate inhibits neutrophil chemotaxis induced by B4 and the complement protein C5a administration inside an air pouch. These results indicated that the anti-inflammatory activity of methotrexate observed in clinical trials with arthritis

1987: The air pouch model was used to study the effect of inflammation over pH. The author concluded that inflammation slightly decreases pH (about 0.5 units) and suggests that the pH

1987: The effect of a fish oil-supplemented diet over inflammation and immune response was studied using a rat air pouch model. The rats were given 500 mg/kg/day of eicosapentaenoic acid and 333 mg/kg/day of docosahexaenoic acid, and control groups received water, oleic acid, or safflower oil for a period of 50 days. After this period, acute and chronic inflammation responses were induced and evaluated in an air pouch model with bovine serum albumin antigen. The results showed that the fish oil-supplemented diet decreases prostaglandin E2 and leukotriene B4 and increases leukocyte infiltration during the chronic inflammation state. 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

1987: The tumor targeting the potential of liposomes encapsulating the radioisotope Ga-67 and an antibody against DLAA (Dalton's lymphoma-associated antigen) was evaluated in an air pouch model. The results showed that anti-DLAA improves Ga-67 uptake by tumor cells and that negative and neutral charge liposomes increase the accumulation of GA-67 to tumor tissue as compared to positively charged liposomes. Liposomes with Ga-67 but without anti-DLAA had minimal accumulation in tumor tissue and maximal accumulation in the liver and

response, by inhibition of leukotriene B4 and prostaglandin E2 synthesis [42].

exchange [36].

spleen [43].

established with a croton oil injection [37].

estrogen-dependent mammary cancers [38].

the most sensitive to assess the effect of steroids [39].

variation could affect the effect of anesthetics [41].

patients may be due to neutrophil chemotaxis inhibition [40].

1974: The healing of tissues damaged by X-ray radiation was studied using the air pouch model. The measurement of radiation damaged was based on the DNA content of the granuloma tissue and the volume of blood produced by angiogenesis and by microcolony count. It was concluded that a single high dose of local radiation (2000–4000 rads) destroys the inner layer of rapidly growing cells, while outer layers of differentiated cells show less damage [29].

1976: Differences between the early and late stages of inflammation were studied in a carrageenan inflammation air pouch model. The results showed that vascular permeability is increased by histamine injection on the early stage, while prostaglandins increase vascular permeability on the chronic stage [30].

1976: Hypersensitivity to tobacco was studied in a mouse model. To determine the involvement of mast cell response, an air pouch was induced on the back of mice, tobacco extract was administered into the pouch, and the tissue was examined microscopically to determine the presence of degranulated and non-degranulated mast cells. It was concluded that mice serve as a good model to study tobacco hypersensitivity and that mast cell is sensitized by tobaccospecific IgG and IgE [31].

1977: The air pouch model with carrageenan was used to evaluate the effect of flurbiprofen, a nonsteroidal anti-inflammatory drug, on prostaglandin production and leucocyte migration. It was concluded that optimal doses of flurbiprofen may inhibit prostaglandin synthesis and polymorphonuclear leucocyte migration into the site of inflammation [32].

1979: It was observed that oxygenation of tumor tissues occurred after X-ray radiation in some tumors; this phenomenon was termed reoxygenation, and it was believed to affect cancer cell survival. Reinhold, Blachiewicz, and Berg-Blok modified the air pouch model to obtain a "sandwich chamber" on the back of rats, to evaluate the oxygenation effect on tumors with or without the previous radiation. They concluded that oxygen levels in the tumor microenvironment of rhabdomyosarcoma BA 1112 can temporarily increase after 20 Gy radiation [33].

1980: N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (BP) were administered into air pouches of adult male rats. Fort-eight hours later, the granulation tissue was excised, and the density of mutated cells resistant to 6-thioguanine was determined in vitro. There was a dose-dependent increase in the mutation rate for both compounds [34].

1980: L. van den Boocaard studied the antitumor effect of heat and irradiation combined. He induced an air pouch on the back of rats and inoculated tumor cells inside the pouch. Then, the dorsal skin of the rat was immersed in a water bath at 42°C, and the air pouch prevented the excessive heating of the body, while tumor was about the same as the water bath. Tumor heating was performed for 2 hours 1 day before irradiation. L. van den Boocaard concluded that the combined therapy induced tumor regression and a longer tumor-free interval [35].

1982: An air pouch was formed on the back of rats to induce rapidly proliferating granuloma tissue. Two days later the test compounds, 5-bromo-2′-deoxyuridine (BrdU), mitomycin C (MMC), colchicine, and cyclophosphamide (CP), were administered inside the pouch; 24 hours later, the tissue was excised, and sister chromatid exchange frequencies were determined. It was concluded that mitomycin C and cyclophosphamide induce sister chromatid exchange [36].

least 6 months. This heterotypic bladder in the rat was described by the authors as the first

1974: The healing of tissues damaged by X-ray radiation was studied using the air pouch model. The measurement of radiation damaged was based on the DNA content of the granuloma tissue and the volume of blood produced by angiogenesis and by microcolony count. It was concluded that a single high dose of local radiation (2000–4000 rads) destroys the inner layer of rapidly growing cells, while outer layers of differentiated cells show less damage [29]. 1976: Differences between the early and late stages of inflammation were studied in a carrageenan inflammation air pouch model. The results showed that vascular permeability is increased by histamine injection on the early stage, while prostaglandins increase vascular

1976: Hypersensitivity to tobacco was studied in a mouse model. To determine the involvement of mast cell response, an air pouch was induced on the back of mice, tobacco extract was administered into the pouch, and the tissue was examined microscopically to determine the presence of degranulated and non-degranulated mast cells. It was concluded that mice serve as a good model to study tobacco hypersensitivity and that mast cell is sensitized by tobacco-

1977: The air pouch model with carrageenan was used to evaluate the effect of flurbiprofen, a nonsteroidal anti-inflammatory drug, on prostaglandin production and leucocyte migration. It was concluded that optimal doses of flurbiprofen may inhibit prostaglandin synthesis and

1979: It was observed that oxygenation of tumor tissues occurred after X-ray radiation in some tumors; this phenomenon was termed reoxygenation, and it was believed to affect cancer cell survival. Reinhold, Blachiewicz, and Berg-Blok modified the air pouch model to obtain a "sandwich chamber" on the back of rats, to evaluate the oxygenation effect on tumors with or without the previous radiation. They concluded that oxygen levels in the tumor microenvironment of rhabdomyosarcoma BA 1112 can temporarily increase after 20 Gy radiation [33]. 1980: N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (BP) were administered into air pouches of adult male rats. Fort-eight hours later, the granulation tissue was excised, and the density of mutated cells resistant to 6-thioguanine was determined in vitro.

There was a dose-dependent increase in the mutation rate for both compounds [34].

1980: L. van den Boocaard studied the antitumor effect of heat and irradiation combined. He induced an air pouch on the back of rats and inoculated tumor cells inside the pouch. Then, the dorsal skin of the rat was immersed in a water bath at 42°C, and the air pouch prevented the excessive heating of the body, while tumor was about the same as the water bath. Tumor heating was performed for 2 hours 1 day before irradiation. L. van den Boocaard concluded that the combined therapy induced tumor regression and a longer tumor-free interval [35]. 1982: An air pouch was formed on the back of rats to induce rapidly proliferating granuloma tissue. Two days later the test compounds, 5-bromo-2′-deoxyuridine (BrdU), mitomycin C (MMC), colchicine, and cyclophosphamide (CP), were administered inside the pouch;

polymorphonuclear leucocyte migration into the site of inflammation [32].

step to study the implantation, progression, and spread of bladder cancer [28].

permeability on the chronic stage [30].

specific IgG and IgE [31].

176 Cell Culture

1983: The carcinogenic potential of procarbazine was evaluated in an air pouch model in rats. It was concluded that high doses of procarbazine (300 mg/Kg) injected into the subcutaneous tissue do not induce tumors, unless an inflammatory environment had been previously established with a croton oil injection [37].

1984: An air pouch model was induced beneath the mammary fat pad of Wistar rats and injected with 20 milligrams of DMBA (7,12-dimethylbenz[a]anthracene). This technique allowed the induction of localized, transplantable, and estrogen-dependent adenocarcinomas in 67–80 days as compared to 150–180 days required by other techniques. The authors suggested that this model might be useful to study the biochemical mechanisms involved in estrogen-dependent mammary cancers [38].

1986: Sedgwick and Lees compared three different acute inflammation models: a 6-day air pouch, polyester sponge, and pleurisy. Overall, they concluded that the air pouch model was the most sensitive to assess the effect of steroids [39].

1987: Pretreatment of mice with methotrexate inhibits neutrophil chemotaxis induced by B4 and the complement protein C5a administration inside an air pouch. These results indicated that the anti-inflammatory activity of methotrexate observed in clinical trials with arthritis patients may be due to neutrophil chemotaxis inhibition [40].

1987: The air pouch model was used to study the effect of inflammation over pH. The author concluded that inflammation slightly decreases pH (about 0.5 units) and suggests that the pH variation could affect the effect of anesthetics [41].

1987: The effect of a fish oil-supplemented diet over inflammation and immune response was studied using a rat air pouch model. The rats were given 500 mg/kg/day of eicosapentaenoic acid and 333 mg/kg/day of docosahexaenoic acid, and control groups received water, oleic acid, or safflower oil for a period of 50 days. After this period, acute and chronic inflammation responses were induced and evaluated in an air pouch model with bovine serum albumin antigen. The results showed that the fish oil-supplemented diet decreases prostaglandin E2 and leukotriene B4 and increases leukocyte infiltration during the chronic inflammation state. 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].

1987: The tumor targeting the potential of liposomes encapsulating the radioisotope Ga-67 and an antibody against DLAA (Dalton's lymphoma-associated antigen) was evaluated in an air pouch model. The results showed that anti-DLAA improves Ga-67 uptake by tumor cells and that negative and neutral charge liposomes increase the accumulation of GA-67 to tumor tissue as compared to positively charged liposomes. Liposomes with Ga-67 but without anti-DLAA had minimal accumulation in tumor tissue and maximal accumulation in the liver and spleen [43].

1988: Bottomley, Griffiths, Rising, and Steward implanted a rat cartilage wrapped in cotton inside an air pouch model in mice. 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].

growth inhibition and increased survival were observed particularly in the LT + UVA radiation group. The results suggest that LT is probably more effective than not encapsulated ana-

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2009: An air pouch model was induced to determine if interleukin-17 could initiate an inflammatory response. It was concluded that interleukin-17 cannot start an inflammatory response,

2009: Kourelis et al. used the air pouch model for the evaluation of early immune response. This group determined the immunoregulatory properties of *Lactobacillus paracasei* subsp. *paracasei*

2013: The air pouch model has also been validated for nanoparticle evaluation by Vandooren et al. This group evaluated biocompatibility, toxicity, and inflammatory and adaptive immunological response to nanoparticles designed for nanomedicine. They reported that this tech-

2014: Eteraf-Oskouei et al. evaluated the antiangiogenic effect of honey in vivo. This group induced an inflammation air pouch model in Wistar rats, by injecting 1 milliliter of carrageenan (1%) on day 6. 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

2015: Another report published by Eteraf-Oskouei et al. evaluated the antiangiogenic effect of *Ficus carica* leaf extract. The volume of exudates, the cell number, and TNFα, PGE2, and VEGF levels contained in carrageenan air pouches were measured, and for angiogenesis determination, they measured hemoglobin quantity. The extract significantly decreased the volume of exudate and leukocyte accumulation and levels of TNFα, PGE2, and VEGF; it also inhibited angiogenesis [57]. 2015: A study evaluated the role of interleukin-17 in invasive breast cancer tumor pathogenesis. Air pouches were created on the back of mice, and 4 t1 or 67NR supernatants (metastatic and nonmetastatic murine mammary cancer cell lines, respectively) were injected into the pouches. 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-17 were determined by flow cytometry analysis. From this experiment

2018: Our group adapted the air pouch model for the screening of antitumor properties of the bio-compound IMMUNEPOTENT CRP. We induced the air pouch model, inoculated L5178Y-R cancer cells, and determined if our compound interfered with tumor implantation.

In our opinion the air pouch model is a valuable technique for the evaluation of compounds with antitumor and tumor-preventive and/or tumor-chemopreventive properties. In **Table 1**, we summed the main advantages and disadvantages of the air pouch model for cancer as

cells were significantly higher in the

might be useful in the treatment of granulomatous inflammatory conditions [56].

tase particles of titanium dioxide for the treatment of bladder cancer [52].

but it is able to increase inflammation in its early stages [53].

nique yields reproducible data, with three mice per test [55].

it was concluded that interleukin-17 producing CD3+

compared to in vitro cell culture.

group treated with the 4 T1 metastatic cancer cell line supernatant [58].

We concluded that our bio-compound has antitumor properties [59].

B112, DC205, DC215, and DC412 strains [54].

1989: The distribution of latex microspheres into inflamed tissues and inflammatory exudate was investigated. 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].

1996: Appleton et al. evaluated the role of vascular endothelial growth factor in inflammationmediated angiogenesis with the air pouch model. They concluded that the vascular endothelial growth factor may be an important regulator of angiogenesis during the inflammation process [46].

1999: The air pouch model was used to evaluate the chemopreventive activity of celecoxib and indomethacin. Briefly, hairless mice were fed with celecoxib or indomethacin, and air pouches were exposed to ultraviolet light. Celecoxib and indomethacin prevented tumor formation in 89% and 78% of the cases, respectively [47].

2000: The activation of NF-κB in the carrageenan air pouch model was studied and also the effect of dexamethasone over NF-κB activation. Results showed that NF-κB activation starts on the first day of inflammation and increases as inflammation progresses and decreases with dexamethasone treatment [48].

2000: Hooper et al. evaluated leukocyte populations, oxygen reactive species release, and phagocytosis, in response to the biomaterials poly(tetrafluoroethylene) (ePTFE), silicone, low-density polyethylene (LDPE), poly(L-lactic acid) (PLLA), poly(desaminotyrosyl-tyrosine ethyl carbonate) [poly(DTE carbonate)], and poly(desaminotyrosyl-tyrosine benzyl carbonate) [poly(DTBzl carbonate)]. Poly(DTE carbonate), ePTFE, LDPE, or poly[DTBzl carbonate increased the levels of superoxide anions inside the pouches. The authors stated that the air pouch method is a highly sensitive method to test the response of inflammatory cells to biomaterials [49].

2001: Retroviral vectors expressing a human interleukin-1 receptor antagonist and a soluble tumor necrosis factor receptor, where injected inside an air pouch model of inflammation. The pouch tissues and exudates were collected after 48 or 72 hours. 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].

2004: An air pouch model was used to determine that acute neutrophilic inflammation in response to urate crystals is dependent of chemokines that bind the mIL-8RH receptor, the mice homolog of CXCR-2 chemokine 8 receptor [51].

2007: A study evaluated the antitumor effect of liposome-encapsulated anatase particles of titanium dioxide (LT). The air pouch was inoculated with NBT-II bladder cancer cells, to simulate bladder cancer, and treated with LT injections followed by UVA radiation. Tumor growth inhibition and increased survival were observed particularly in the LT + UVA radiation group. The results suggest that LT is probably more effective than not encapsulated anatase particles of titanium dioxide for the treatment of bladder cancer [52].

1988: Bottomley, Griffiths, Rising, and Steward implanted a rat cartilage wrapped in cotton inside an air pouch model in mice. 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

1989: The distribution of latex microspheres into inflamed tissues and inflammatory exudate was investigated. Briefly, an air pouch model with carrageenan was induced in rats, and microspheres were administrated either orally or intravenously. The authors concluded that

1996: Appleton et al. evaluated the role of vascular endothelial growth factor in inflammationmediated angiogenesis with the air pouch model. They concluded that the vascular endothelial growth factor may be an important regulator of angiogenesis during the inflammation

1999: The air pouch model was used to evaluate the chemopreventive activity of celecoxib and indomethacin. Briefly, hairless mice were fed with celecoxib or indomethacin, and air pouches were exposed to ultraviolet light. Celecoxib and indomethacin prevented tumor for-

2000: The activation of NF-κB in the carrageenan air pouch model was studied and also the effect of dexamethasone over NF-κB activation. Results showed that NF-κB activation starts on the first day of inflammation and increases as inflammation progresses and decreases with

2000: Hooper et al. evaluated leukocyte populations, oxygen reactive species release, and phagocytosis, in response to the biomaterials poly(tetrafluoroethylene) (ePTFE), silicone, low-density polyethylene (LDPE), poly(L-lactic acid) (PLLA), poly(desaminotyrosyl-tyrosine ethyl carbonate) [poly(DTE carbonate)], and poly(desaminotyrosyl-tyrosine benzyl carbonate) [poly(DTBzl carbonate)]. Poly(DTE carbonate), ePTFE, LDPE, or poly[DTBzl carbonate increased the levels of superoxide anions inside the pouches. The authors stated that the air pouch method is a highly sensitive method to test the response of inflammatory cells to bio-

2001: Retroviral vectors expressing a human interleukin-1 receptor antagonist and a soluble tumor necrosis factor receptor, where injected inside an air pouch model of inflammation. The pouch tissues and exudates were collected after 48 or 72 hours. 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

2004: An air pouch model was used to determine that acute neutrophilic inflammation in response to urate crystals is dependent of chemokines that bind the mIL-8RH receptor, the

2007: A study evaluated the antitumor effect of liposome-encapsulated anatase particles of titanium dioxide (LT). The air pouch was inoculated with NBT-II bladder cancer cells, to simulate bladder cancer, and treated with LT injections followed by UVA radiation. Tumor

microspheres could be used as a vehicle for inflammation-targeted treatment [45].

predict therapeutic efficacy in men [44].

mation in 89% and 78% of the cases, respectively [47].

mice homolog of CXCR-2 chemokine 8 receptor [51].

dexamethasone treatment [48].

process [46].

178 Cell Culture

materials [49].

evaluate gene therapy [50].

2009: An air pouch model was induced to determine if interleukin-17 could initiate an inflammatory response. It was concluded that interleukin-17 cannot start an inflammatory response, but it is able to increase inflammation in its early stages [53].

2009: Kourelis et al. used the air pouch model for the evaluation of early immune response. This group determined the immunoregulatory properties of *Lactobacillus paracasei* subsp. *paracasei* B112, DC205, DC215, and DC412 strains [54].

2013: The air pouch model has also been validated for nanoparticle evaluation by Vandooren et al. 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].

2014: Eteraf-Oskouei et al. evaluated the antiangiogenic effect of honey in vivo. This group induced an inflammation air pouch model in Wistar rats, by injecting 1 milliliter of carrageenan (1%) on day 6. 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].

2015: Another report published by Eteraf-Oskouei et al. evaluated the antiangiogenic effect of *Ficus carica* leaf extract. The volume of exudates, the cell number, and TNFα, PGE2, and VEGF levels contained in carrageenan air pouches were measured, and for angiogenesis determination, they measured hemoglobin quantity. The extract significantly decreased the volume of exudate and leukocyte accumulation and levels of TNFα, PGE2, and VEGF; it also inhibited angiogenesis [57].

2015: A study evaluated the role of interleukin-17 in invasive breast cancer tumor pathogenesis. Air pouches were created on the back of mice, and 4 t1 or 67NR supernatants (metastatic and nonmetastatic murine mammary cancer cell lines, respectively) were injected into the pouches. 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-17 were determined by flow cytometry analysis. From this experiment it was concluded that interleukin-17 producing CD3+ cells were significantly higher in the group treated with the 4 T1 metastatic cancer cell line supernatant [58].

2018: Our group adapted the air pouch model for the screening of antitumor properties of the bio-compound IMMUNEPOTENT CRP. We induced the air pouch model, inoculated L5178Y-R cancer cells, and determined if our compound interfered with tumor implantation. We concluded that our bio-compound has antitumor properties [59].

In our opinion the air pouch model is a valuable technique for the evaluation of compounds with antitumor and tumor-preventive and/or tumor-chemopreventive properties. In **Table 1**, we summed the main advantages and disadvantages of the air pouch model for cancer as compared to in vitro cell culture.


**Table 1.** Comparison between the *in vitro* and the *in vivo* air pouch model cell line culturing and drug discovery.
