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).