Preclinical CRO offering Xenograft Studies

Applications of Xenografting

Scientific Applications of Xenografts

Since the first reported successful allograft in animals, performed by Samuel Bigger to a blind gazelle that needed a new transparent cornea (Bigger, 1837), and the first successful corneal transplant in humans, performed by Eduard Zirm (1906), techniques have been continuously improving, and many types of transplants are now possible (Nature Biotechnology, 2000).

Holan and Krulova (2013) described the role of “killer” macrophages, also known as M1, activated by Th1 or Th17 cytokines during transplant rejection, and the role played by the “healer” macrophages (M2), activated by some Th2 cytokines, in healing process. At the place of graft rejection, macrophages, CD4+ T cells, and CD8+ T cells are frequent. The macrophages produce nitric oxide (NO), a toxic molecule detected during allograft rejection, and the process is modulated by the nitric oxide synthase / arginase ratio. Both enzymes utilize L-arginine as a substrate, and their expression is regulated by Th1 and Th2 cytokines. While the nitric oxide levels increase during allograft rejection, the xenograft rejection and the allograft healing are characterized by a high level of arginase, and consequently, a low level of NO.

Jardim-Perasse et al. (2014) used xenograft models of breast cancer which were daily injected with melatonin, and studied the tumor growth. During a 21-day test, they observed growth reduction in treated mice compared to the controls, and the reduction of the tumor was recorded for one animal.  Lu et al. (2013) developed a quantitative analysis method for the polyphenolic ketone rottlerin from the plasma and tumors of xenograft murine models, after their previous demonstration of tumor growth reduction due to the above-mentioned compound. The mice were sacrificed after a 6-week diet with 0.012% rottlerin, and the ketone level was determined by reverse-phase HPLC-DAD method, proving its effective cellular absorption, a promising result for cancer treatment.

Leonard et al. (2014) studied the cytotoxic effect of cytarabine on xenograft models in Childhood Acute Myeloid Leukimia treatment. They proved that decitabine enhances the tumor reduction when used together with cytarabine, by determining the bone marrow CD45+/CD33+ cell population.

Benefits of Xenografts

Our in vivo xenograft CRO services ensure that you get the best, most accurate results in the least amount of time possible. Xenografts are the perfect way to deliver on this promise because they are easy to use, comparatively inexpensive and the results are always reproducible. As with all biological work in model organisms, xenografts also have some drawbacks in that they may be unrepresentative of the genetics and histology of the human tumors and as such, sometimes may not provide an accurate prediction of the success of a particular therapy.

The Success of Xenografts

The key advantages of utilizing human tumor xenografts outweigh the disadvantages. Xenografts have these successes:

  • Represent the complexity of genetic and epigenetic abnormalities found commonly in human population
  • They can assist in the creation of personalized molecular therapies
  • Multiple therapies can be tried on a single tumor biopsy
  • The stroma from the human tumor microenvironment can be incorporated into the xenograft to more completely imitate the human tumor micro-environment

Xenograft Models and Their Advantages

Heterotopic tumor xenograft models have been used for more than 30 years. These are the most widely used preclinical mouse models because of the following advantages for cancer research:

  • Fast results
  • Inexpensive
  • Models can be reproduced easily
  • Considered adequate as a preclinical test of anti-cancer drugs
  • There is visual evidence that mice have the tumors prior to the administration of test therapies
  • Provide a visual method of reviewing tumor reaction or increase over a period of time

Orthotopic tumor xenografts fill in gaps where the heterotopic tumor models fail. These go one step further and employ the following advantages for researchers:

  • A relevant site is used to test the tumor-host interactions
  • Disease-relevant metastases can be observed
  • Researchers have the ability to study site-specific effects of therapy

Why Use Human Tumor Xenografts?

There is no doubt that the generation of tumor xenografts can be labor intensive, expensive and requires longer healing and recovery time. Even so, tumor models are becoming the chosen method for cancer research because of their improved clinical relevance. Using human tumor xenografts provides researchers with a novel way to examine the diseases’ therapeutic response to drugs.

How to order PDX xenograft animal testing service? – please visit our Patient Testing webpage


Xenotransplantation has allowed the study of many types of cancer, including bladder, breast, ovarian, and pancreatic, among others. Cancer models available at Altogen Labs include:

  • Pancreatic Cancer: The PANC-1 cell line can be used to model human pancreatic carcinoma in a murine model. In one study, a hind leg xenograft of PANC-1 was used to test an engineered vaccinia virus as a potential treatment for pancreatic carcinoma. In addition, luciferase-expressing PANC-1 cells have been used as an orthotopic xenograft in an imaging study of the effects of ionizing radiation on pancreatic tumors.
  • Lung Cancer: The NCI-H292 cell line can be used to model non-small cell lung cancer for the evaluation of novel therapeutics. An example of this use can be seen in this study, where NCI-H292 and several other cell lines were used to evaluate the efficacy of a monoclonal antibody treatment, when compared with or combined with radiation, chemotherapy or chemoradiation treatments.

Other Drug Testing

  • inflammation
  • diabetes
  • immunology
  • obesity
  • pain research

Standard Xenograft Protocol For Pancreatic Cancer Lines

Preparation of pancreatic cancer cell lines into single cell suspensions

  1. Rinse cell culture plate twice with sterile PBS.
  2. Add 2 ml of 1X trypsin-EDTA (0.05%) to plate or enough to just cover surface area of cells. Place back in 37°C incubator.
  3. Three minutes after the addition of trypsin, examine cell adherence to surface of cell culture plate or flask. Lightly tap the cell culture plate or flask if majority of cells have been released. If many cells remain adherent, place back in 37°C incubator for an additional 3 minutes, lightly tapping cell container as needed.
  4. Once most cells (>98%) have detached from the plate, add sterile culture media 5:1 to volume of trypsin added to culture flask. Gently pass cell suspension through 10ml pipette 5 times to reduce cell clumping and to remove remaining adherent cells.
  5. Collect cells into 15ml or 50ml centrifuge tube, depending on the volume of cell solution collected. Proceed to centrifuge cells at 1200rpm for 5 minutes, resulting in the formation of a cell pellet. Wash cell pellet once with sterile PBS and re-suspend pellet in sterile culture media.
  6. Quantitate cells using a hemacytometer or cell counter. Adjust cell concentration to 1 million cells/ml with sterile culture media.
  7. Divide into aliquots of 1 × 106 cells (10 ml) and again centrifuge at 1200rpm for 5 minutes. Thoroughly re-suspend pellet in 20mL of sterile Hank’s balanced salt solution (HBSS) containing 1% serum-free matrigel by volume (4°C), bringing cell concentration to 500,000cells per 100ul (final cell inoculum).
  8. The concentration of cells in the prepared cell solution may be altered depending on the desired cell inoculum, site of injection, and specific experimental objectives. Regardless, due to technical considerations, one should prepare at least twice the volume of cell solution required for experiments.
  9. Keeping cell solution on ice, aspirate into an open 1.0 ml syringe and cap with a 25 gauge needle.
  10. Proceed to heterotopic or orthotopic cell injection. It is important to maintain 1% matrigel-cell solution on ice at all times to prevent premature gel formation.

Orthotopic implantation of pancreatic cancer cells

  1. Prepare pancreatic cancer cell solution (from cell lines or tumor) to a concentration of 500,000 cells per 100µl in Hank’s balanced salt solution containing 1% matrigel. Leave on ice with occasional vortex agitation.
  2. Anesthetize recipient nude or NOD/SCID mice.
  3. Shave left abdominal/flank region of mice with clippers and place mice on their right side. Paint left sides of mice from the base of the neck to tail with a 70% ethanol solution.
  4. Proceed with animal surgery and cell implantation.
  5. Identify silhouette of spleen through intact, shaved skin. Pick up skin with forceps and make a 1.2 cm incision with sterile micro-scissors slightly medial to splenic silhouette.
  6. Grasp underlying muscle with forceps and lift to enter the abdominal cavity without injury to underlying organs. Extend muscle incision with micro-scissors to 0.75cm.
  7. Using a pair of blunt-nose forceps, gently grasp the tip of the pancreatic tail and externalize pancreas/spleen in a lateral fashion, exposing the entire pancreatic body and spleen.
  8. Remove syringe containing cell suspension (cell line or xenograft-derived) from ice and vortex agitate to disrupt cell clumps/cluster.
  9. While gently retracting pancreas laterally, insert needle into the tail of pancreas and pass into the pancreatic head region. Slowly inject 100µl of cell solution while withdrawing needle to mid-body of pancreas. Remove needle from pancreas and observe needle tract for leak/bleeding.
  10. Leave pancreas externalized and untouched for 2 minutes for matrigel to solidify while inspecting pancreas for vascular injury and/or leakage of cell solution. Gently internalize pancreas/spleen with blunt forceps and close abdominal muscle layer with interrupted stitches (3-0 silk suture). Close overlying skin with a second set of interrupted stitches (3-0 silk suture) and apply antibiotic ointment.
  11. Recover animals from anesthetic.

Heterotopic implantation of pancreatic cancer cells

  1. Anesthetize immunocompromised nude or NOD/SCID mice.
  2. If required, shave desired injection site and place mouse on its contralateral side. Depending on the location of cell implantation, paint corresponding side of mouse with 70% ethanol solution.
  3. Gently agitate syringe containing cell suspension to disrupt cell clumps/clusters.
  4. Identify desired location of cell implantation. While gently grasping skin with forceps for counter-traction, penetrate skin with the needle tip approximately a needle’s length from desired injection site. Direct needle to desired implantation site while passing the needle parallel to animal body.
  5. Once the needle is confirmed to be in the subcutaneous space, slowly inject 150 µl. Confirm successful cell injections by presence of bulla.
  6. Withdraw needle slowly from tract and monitor for 2 minutes for gross leakage.
  7. Recover mice from the effects of anesthetic.