2.5.1.3 Ehrlich Ascites carcinoma
On the day 0, 2x105 tumour cells are injected intraperitoneally to cause tumours in test animals. The pharmacological therapy starts 24 hours after the tumour has been inoculated. After the predetermined intervals, the animals are killed, and the peritoneal fluid is harvested. Several saline washes are used to repeatedly remove tumour cells from the peritoneal cavity. The survival time assay might include more animal species. The following measurements are used for evaluation: peritoneal fluid volume, tumour cell viability in peritoneal fluid, packed cell volume (PCV) in peritoneal fluid, and percentage increase in survival time of drug-treated mice. In order to assess how well test drugs affect tumours, haematological, biochemical, and tumour cell morphology are also studied(Gomes et al., 2010).
Cell densities of the cancer can range from 25 to 100 million per millilitre of ascitic fluid as it spreads through the peritoneal fluid. This model is frequently used for initial screening because it forecasts widespread antitumor activity. A modified solid tumour model is created by subcutaneously injecting 4x106 tumour cells into the animal’s flank. In 14 days, the tumour grew to a diameter of 12 mm. The two main methods of evaluation are tumour volume and histological examination of tumours(Fahim et al., 2003).
2.5.2 Xenograft models: The tumour models that are most closely related to clinical illness should be created using human-derived transplantable tumours. Such human tumours might cause mice to develop severe immunological rejection, though. Animals with severe combined immunodeficiency (NOD-SCID) or athymic (nude) mice are both used in this study. The transplanted foreign material has no effect on these animals’ immune systems. Before athymic mice were available, immune compromised mice brought on by irradiation, thymectomy, or steroids were used for transplantation.
The first nude mice, which spontaneously appeared in an enclosed colony of albino mice in a laboratory in Glasgow, Scotland, were described by Isaacson and Cattanach as having no fur. On chromosome 11, there is an autosomal recessive mutant gene (nu, which stands for nude) that causes hair loss, sluggish growth, short lifespan, and low fertility. The absence of a thymus in nu/nu athymic mice results in a deficiency in T cells, whereas nu/1 mice, who receive a thymus from their heterozygous mother, do. However, these animals’ B cell function is normal, and their natural killer cell activity is increased(Rygaard and Povlsen, 1969).
The success of xenografting human tumours into nude mice and the ability to maintain the histologic and biologic identity of tumours in vivo over several passages have both revolutionised cancer research in various ways. Tumor cell lines can be implanted into naked mice using subcutaneous, intraperitoneal, intravenous, intracranial, intrasplenic, renal subcapsular, and a new orthotopic model utilising site-specific organ injection.
The human tumour cells show kinetic alterations when implanted into nude mice. Most of the time, the doubling time is faster than the original tumour, and it gets faster in successive passes. Many xenografted human cancers retain their original morphologic and metabolic properties despite this. As a result, human tumour xenografts are the backbone of cancer therapy development(Friess et al., 2005).
2.5.2.1 Subcutaneous implantation: Because of the convenience of the procedure, this route is thought to be the most accessible one for the transplantation of human tumours into nude mice. This technique has been selected by the National Cancer Institute as the main in vivo test for its drug research and screening programme. Usually, a suspension of tumour cells is injected into the animal’s flank (about 106 to 107 cells per animal). The length of time it takes for a tumour to grow can vary depending on the cell line that is used. With subcutaneous xenografts, metastases and invasion of neighbouring tissues are uncommon(Navale, 2013).
2.5.2.2 Renal Subcapsular (RSC) Assay: The Bogden and colleagues first presented this methodology in 1978. The cells are injected into a naked mouse as a 1 mm tumour fragment under the kidney capsule. These tumours have the advantage of retaining the true morphologic, functional, and growth traits of the original tumour, including cell-cell contact and tumour spatial connection. In doing so, they provide a more accurate representation of the features of human tumour metastatic features. Animal survival tests, clonogenic tests, and growth tests are appropriate evaluation techniques.
In contrast to the subcutaneous xenograft assay, the renal subcapsular assay has a relatively short and constant time between the injection of the tumour and the appearance of a graphically palpable mass. Tumors are routinely examined over a six-day period. In situations where a quick in vivo experiment is required, this model can be helpful. It has many benefits, but because the kidney’s subcapsular region is not immune-privileged, it is not the ideal model. It’s possible that the original tumour or another factor led to the proliferation of numerous lymphocytes that have infiltrated the tumour in this area. However, the orthotopic model for renal cell carcinoma may be helpful(Navale, 2013).
2.5.2.3 Intraperitoneal Microencapsulated Tumor Assay: Due to the RSC’s limitations and limited ability to adapt to slow-growing malignancies, alternative short-term in vivo assays have been created. One of these uses microencapsulation technology: the microencapsulated tumour assay. The semi-permeable gels that can be manufactured into 0.05 to 1 mm in size microcapsules enclose tumour cells. These microcapsules are inoculable in experimental animals’ peritoneal area. Around 600 microcapsules are injected into the peritoneum in normal test settings using the mouse. The capsule half-permeability protects the tumour cells from immune cytotoxicity through the host cells and does not necessarily require anthymical (nude) mice. It also enhances the circulation and reaching of tumour cells by nutritional and systemic cytotoxic substances. By rehabilitating and comparing the number of live tumour cells in treated versus control animals, it is possible to assess how effective the anti-cancer effect is. The microencapsulation assay is simple, efficient, and reasonably priced. It uses fewer mice than the subcutaneous implanted tumour experiment. After being exposed to medication doses that would be present in vivo, tumour cells are tested. Furthermore, the method can be applied to the majority of solid tumours and uses immune competent mice, unlike the subcutaneous transplanted tumour experiment. Multiple tumours in the same mouse can be evaluated concurrently. In order to follow up, on first therapy leads that pass the in vitro screening method, the NCI screening programme uses the micro encapsulated tumour assay as an in vivo second-line screen(Navale, 2013).
2.5.2.4 Orthotopic Xenograft Model: Transgenic tumour models and subcutaneously developing human tumours in immune compromised mice do not accurately replicate human clinical cancers because, when implanted heterotopically, they lose metastatic potential and change medication sensitivity(Fidler, 1986).
A system of tumour cells transplanted at the site of the organ of genesis is the orthotopic xenograft model. When the SOI (surgical orththotopic implantation) models were compared to transgenic mouse cancer models, the SOI models were found to be more relevant to clinical metastatic cancer(Shapiro et al., 1979). This organ-specific location is thought to offer the tumour cells with the best conditions for growth and development. This methodology has yet to be widely adopted by the NCI drug-screening programme due to its high cost and novelty(Brigelius-Flohé and Flohé, 2020).