Patient-derived tumor xenograft

Patient derived tumor xenografts (PDTX) are created when cancerous tissue from a patient’s primary tumor is implanted directly into an immunodeficient mouse. PDTX models are providing solutions to the challenges that researchers face in cancer drug research such as positive tumor responses in mouse models but not translating over when the study is implemented in humans. As a result, PDTX cancer models are becoming popular models to use in cancer drug research.

Methods of tumor xenotransplantation

Several types of immunodeficient mice can be used to establish PDTX models: athymic nude mice, severely compromised immune deficient (SCID) mice, nonobese diabetic (NOD)-SCID mice, and recombination-activating gene 2 (Rag2)-knockout mice.[1] The mice used must be immunocompromised to prevent transplant rejection. The NOD-SCID mouse is considered more immunodeficient than the nude mouse, and therefore is more commonly used for PDTX models because the NOD-SCID mouse does not produce Natural Killer cells.[2]

When human tumors are resected, necrotic tissues is removed and the tumor is either mechanically sectioned into smaller fragments or chemically digested or physically manipulated into a single-cell suspension. There are both advantages and disadvantages in utilizing either discrete tumor fragments or single-cell suspensions. Tumor fragments retain cell-cell interactions as well as some tissue architecture of the original tumor, therefore mimicking the tumor microenvironment. Alternatively, single-cell suspension enables scientists to collect an unbiased sampling of the whole tumor, eliminating spatially segregate subclones that are otherwise inadvertently selected during analysis or tumor passaging[3] However, single-cell suspensions subject surviving cells to harsh chemical or mechanical forces that may sensitize cells to anoikis, taking a toll on cell viability and engraftment success.[4]

Unlike creating xenograft mouse models using existing cancer cell lines, there are no intermediate in vitro processing steps before implanting tumor fragments murine host to create a PDTX. The tumor fragments are either be implanted heterotopically or orthotopically of an immunodeficient mouse. Heterotopical implants occur when the tumor fragment is implanted into an area of the mouse unrelated to the original tumor site, generally subcutaneously or subrenal capsular sites.[5] Whereas, scientists tranplant the patient’s tumor tissue and implants the fragments into the corresponding anatomical position in the mouse in an orthotopic transplant. Subcutaneous PDTX rarely produce metastasis in mice, does not simulate the initial tumor microenvironment, and has engraftment rates 40-60%.[5] Subrenal capsular PDTX maintains the original tumor stroma as well as the equivalent host stroma and has an engraftment rate of 95%.[6] Ultimately, the time it takes about 2 to 4 months for the tumor to engraft varying by tumor type, implant location, and strain of immunodeficient mice utilized; engraftment failure should not be declared until at least 6 months.[1]

The first generation of mice receiving the patient's tumor fragments are commonly denoted F0. When the tumor-burden becomes too large for the F0 mouse, researchers passage the tumor over to the next generation of mice. Each generation thereafter is denoted F1, F2, F3…Fn. For drug development studies, expansion of mice after the F3 generation is often utilized after ensuring that the PDTX has not genetically or histologically diverged from the patient’s tumor.[7]

Advantages over cancer cell lines

Cancer cell lines (CCL) are originally derived from patient tumors, but acquire the ability to proliferate within in vitro cell cultures. As a result of in vitro manipulation, CCL that have been traditionally used in cancer research undergo genetic transformations that are not restored when cells are allowed to grow in vivo.[8] Because of the cell culturing process, which includes enzymatic environments and centrifugation, cells that are better adapted to survive in culture are selected, tumor resident cells and proteins that interact with cancer cells are eliminated, and the culture becomes phenotypically homogeneous.[3]

When implanted into immunodeficient mice, CCL do not easily develop tumors and the result of any successfully grown tumor is a genetically divergent tumor unlike the heterogeneous patient tumor.[3] Researchers are beginning to attribute the reason that only 5% of anti-cancer agents are approved by the Food and Drug Administration after pre-clinical testing to the lack of tumor heterogeneity and the absence of the human stromal microenvironment.[9] Specifically, CCL-xenografts often are not predictive of the drug response in the primary tumors because CCL do not follow pathways of drug resistance or the effects of the microenvironment on drug response found in human primary tumors.[9]

Many PDTX models have been successfully established for breast, prostate, colorectal, lung, and many other cancers because there are distinctive advantages when using PDTX over CCL for drug safety and efficacy studies as well as predicting patient tumor response to certain anti-cancer agents.[10] Since PDTX can be passaged without in vitro processing steps, PDTX models allow the propagation and expansion of patient tumors without significant genetic transformation of tumor cells over multiple murine generations.[11] Within PDTX models, patient tumor samples grow in physiological relevant tumor microenvironments that mimic the oxygen, nutrient, and hormone levels that are found in the patient’s primary tumor site.[7] Furthermore, implanted tumor tissue maintains the genetic and epigenetic abnormalities found in the patient and the xenograft tissue can be excised from the patient to include the surrounding human stroma.[12] As a result, numerous studies have found that PDTX models exhibit similar responses to anti-cancer agents as seen in the actual patient who provided the tumor sample.[13] PDTX models are beneficial to use to study therapeutic responses to drugs because multiple therapies can be tested against one biopsy and pre- and post-treatment data can be acquired from the human biopsy and xenograft tissues, potentially sparing a patient from therapies that may not work.[12]

Humanized-xenograft models

One prominent shortcoming of PDTX models is that immunodeficient mice must be used to prevent immune attacks against the xenotransplanted tumor. Therefore, a critical component of the known tumor microenvironment interaction is foregone. As a result, immunotherapies and anti-cancer agents that target the immune system components cannot be studied using PDTX models. Consequently, researchers are beginning to explore the use of humanized-xenograft models. Humanized-xenograft models are created by co-engrafting the patient tumor fragment and peripheral blood or bone marrow cells into a NOD/SCID mouse.[2] The co-engraftment allows for reconstitution of the murine immune system enabling researchers to study the interactions between xenogenic human stroma and tumor environments in cancer progression and metastasis.[14] However, these strategies have yet to be validated for most tumor types and there remains questions over whether the reconstituted immune system will behave in the same way as it does in the patient. For example, the immune system could be 'hyper-activated' due to exposure to mouse tissues in a similar fashion to graft versus host disease.[15] Humanized-xenograft models for acute lymphoblastic leukemia and acute myeloid leukemia have been created.[16]

Clinical relevance

Breast cancer

There have been many advances in breast cancer biology resulting in the classification of different molecular and genetic breast cancer subtypes including triple-negative and HER2-positive subtypes.[7] Oncologists can use a patient’s breast cancer subtype to personalize cancer therapy schedules to better address the patient’s tumor distinct gene-expression profile. Utilizing PDTX triple negative breast cancer models, scientists found that aurora kinase inhibitors slows tumor growth rate and suppresses recurrence in a breast cancer subtype that has a high recurrence rate and poor survivability.[17] Scientists have also found that breast cancer PDTX models are capable of predicting the prognosis of newly diagnosed women by observing the rate of tumor engraftment to determine if the patient tumor is aggressive.[18]

Colorectal cancer

Colorectal PDTX models are relatively easy to establish and the models maintain genetic similarity of primary patient tumor for about 14 generations.[19] In 2012, a study established 27 colorectal PDTX models that did not diverge from their respective human tumors in histology, gene expression, or KRAS/BRAF mutation status.[20] Due to their stability, the 27 colorectal PDTX models may be able to serve as pre-clinical models in future drug studies. Drug resistance studies have been conducted using colorectal PDTX models. In one study, researchers found that the models predicted patient responsiveness to cetuximab with 90% accuracy.[21] Another study identified the amplification of ERBB2 as another mechanism of resistance, and a putative new actionable target in treatments.[22]

Pancreatic cancer

Researchers initially focused on using pancreatic PDTX models for drug studies to improve the process to develop predictive and pharmacodynamics end points for several molecularly targeted therapies.[7] Other studies have been conducted to explore if pancreatic PDTX models can be used to guide the ongoing treatment of advance pancreatic cancer patient by screening multiple drugs to select the drug with most activity as the next line of treatment.[23] Pancreatic PDTX models have enabled researchers to simultaneously run pre-clinical and clinical trial to test the effectiveness of drugs in cancer patients.[24]

Pediatric cancer (neuroblastoma)

Researchers from Lund University, Sweden, have established neuroblastoma PDXs by orthotopic implantation of patient tumor explants into immunodeficient mice.[25] The PDXs retained the genotype and phenotype of patient tumors and exhibited substantial infiltrative growth and metastasis to distant organs including bone marrow. The researchers cultured PDX-derived neuroblastoma cells in vitro and the cells retained tumorigenic and metastatic capacity in vivo. These models will enable identification of compounds effective against metastatic neuroblastoma.[25]

Pediatric Preclinical Testing Program

The Pediatric Preclinical Testing Program (PPTP) is operated by St. Jude Children’s Research Hospital sponsored through a National Cancer Institute research contract. The purpose of the PPTP is to identify more effective treatments for childhood cancers through systematic evaluation of cancer drugs in childhood solid tumors and leukemia models.[26] The PPTP is a collaborative effort between research institutions and pharmaceutical companies to test novel agents against PPTP developed pre-clinical cancer models.[27] The program established panels of childhood cancer xenografts as well as cell lines models to be used for in vivo and in vitro testing, including models for Ewing sarcoma, Wilms tumor, neuroblastoma, glioblastoma, and acute lymphoblastic leukemia. In 2007, in vivo testing on 51 solid tumor and 10 acute lymphoblastic leukemia models established the broad-spectrum activity of vincristine and cyclophosphamide in treating childhood cancers.[28] As of April 2014, there has been 62 peer-review publications releasing data from the project.[26]

Principal investigators

Name Institution
Dr. Peter Houghton, PhD The Research Institute Nationwide Children's Hospital
Dr. E. Anders Kolb, MD A.I duPont Children’s Hospital Wilmington
Dr. Richard Gorlick, MD Children's Hospital at Montefiore Albert Einstein College of Medicine
Dr. Stephen Keir, PhD Preston Robert Tisch Brain Tumor Center Duke University
Dr. John Maris, MD Children's Hospital of Philadelphia
Dr. Richard Lock, PhD Children's Cancer Institute Randwick, Australia
Dr. C. Patrick Reynolds, MD, PhD Texas Tech University Health Science Center Lubbock

Challenges with PDTX model adaptation

There are several challenges that scientists face when developing or using PDTX models in research. For instance not all tumor samples will successfully engraft in the immunodeficient mouse.[10] When engraftment does occur, clinical study protocols are difficult to standardize if engraftment rates vary.[10] There are financial challenges that slow the adaption of PDTX models as the standard for drug development. In the US, the cost to develop PDTX models is not covered by insurance and can potentially cost a patient $25,500 just for doctors to have access to the technology to potentially guide the patient's treatment.[29] Furthermore, the cost just for researchers rise steeply when considering the resources one must utilize to house mice, maintain histopatholigcal cores for frequent testing,[10] and pay for the expertise necessary to perform ex vivo passaging of tumors in mice with high tumor burdens.[2] Therefore, many institutions cannot afford to run PDTX labs because of limited funds.

Commercial companies and contract research organizations

Commercial companies and other contract research organizations are beginning to emerge on the market. As more companies and organizations are formed, the competition may help to lower the costs and provide the expertise needed to develop and maintain PDTX models.

Canada

China

France

Germany

India

United States of America

References

  1. 1 2 Morton CL, Houghton PJ (2007). "Establishment of human tumor xenografts in immunodeficient mice". Nature Protocols (Protocol) 2 (2): 247–50. doi:10.1038/nprot.2007.25. PMID 17406581.
  2. 1 2 3 Siolas D, Hannon GJ (September 2013). "Patient-derived tumor xenografts: transforming clinical samples into mouse models". Cancer Research (Perspective) 73 (17): 5315–9. doi:10.1158/0008-5472.CAN-13-1069. PMC 3766500. PMID 23733750.
  3. 1 2 3 Williams SA, Anderson WC, Santaguida MT, Dylla SJ (September 2013). "Patient-derived xenografts, the cancer stem cell paradigm, and cancer pathobiology in the 21st century". Laboratory Investigation 93 (9): 970–82. doi:10.1038/labinvest.2013.92. PMID 23917877.
  4. ↑ Zvibel I, Smets F, Soriano H (2002). "Anoikis: roadblock to cell transplantation?". Cell Transplantation (Cognizant Communication Publications) 11 (7): 621–30. PMID 12518889.
  5. 1 2 Jin K, Teng L, Shen Y, He K, Xu Z, Li G (July 2010). "Patient-derived human tumour tissue xenografts in immunodeficient mice: a systematic review". Clinical & Translational Oncology (Review) (Springer and FESEO) 12 (7): 473–80. doi:10.1007/s12094-010-0540-6. PMID 20615824.
  6. ↑ Cutz JC, Guan J, Bayani J, et al. (July 2006). "Establishment in severe combined immunodeficiency mice of subrenal capsule xenografts and transplantable tumor lines from a variety of primary human lung cancers: potential models for studying tumor progression-related changes". Clinical Cancer Research 12 (13): 4043–54. doi:10.1158/1078-0432.CCR-06-0252. PMID 16818704.
  7. 1 2 3 4 Tentler JJ, Tan AC, Weekes CD, et al. (June 2012). "Patient-derived tumour xenografts as models for oncology drug development". Nature Reviews. Clinical Oncology (Review) 9 (6): 338–50. doi:10.1038/nrclinonc.2012.61. PMC 3928688. PMID 22508028. Note: open access via PMC; closed via publisher site.
  8. ↑ Daniel VC, Marchionni L, Hierman JS, et al. (April 2009). "A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro". Cancer Research 69 (8): 3364–73. doi:10.1158/0008-5472.CAN-08-4210. PMC 2821899. PMID 19351829.
  9. 1 2 Hutchinson L, Kirk R (April 2011). "High drug attrition rates--where are we going wrong?". Nature Reviews. Clinical Oncology (Editorial) 8 (4): 189–90. doi:10.1038/nrclinonc.2011.34. PMID 21448176.
  10. 1 2 3 4 Malaney P, Nicosia SV, Davé V (March 2014). "One mouse, one patient paradigm: New avatars of personalized cancer therapy". Cancer Letters (Mini-review) 344 (1): 1–12. doi:10.1016/j.canlet.2013.10.010. PMC 4092874. PMID 24157811.
  11. ↑ Reyal F, Guyader C, Decraene C, et al. (2012). "Molecular profiling of patient-derived breast cancer xenografts". Breast Cancer Research 14 (1): R11. doi:10.1186/bcr3095. PMC 3496128. PMID 22247967.
  12. 1 2 Richmond A, Su Y (2008). "Mouse xenograft models vs GEM models for human cancer therapeutics". Disease Models & Mechanisms (Editorial) 1 (2–3): 78–82. doi:10.1242/dmm.000976. PMC 2562196. PMID 19048064.
  13. ↑ Kerbel RS (2003). "Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved". Cancer Biology & Therapy (Review) (Taylor & Francis) 2 (4 Suppl 1): S134–9. PMID 14508091.
  14. ↑ Talmadge JE, Singh RK, Fidler IJ, Raz A (March 2007). "Murine models to evaluate novel and conventional therapeutic strategies for cancer". The American Journal of Pathology (Review) 170 (3): 793–804. doi:10.2353/ajpath.2007.060929. PMC 1864878. PMID 17322365.
  15. ↑ Cassidy JW, Caldas C, Bruna A (July 2015). "Maintaining tumour heterogeneity in patient derived tumour xenografts". Cancer Research (Review) 75 (15): 1–6. doi:10.1158/0008-5472.CAN-15-0727. PMC 4539570. PMID 26180079.
  16. ↑ Meyer LH, Debatin KM (December 2011). "Diversity of human leukemia xenograft mouse models: implications for disease biology". Cancer Research (Review) 71 (23): 7141–4. doi:10.1158/0008-5472.CAN-11-1732. PMID 22088964.
  17. ↑ Romanelli A, Clark A, Assayag F, et al. (December 2012). "Inhibiting aurora kinases reduces tumor growth and suppresses tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts". Molecular Cancer Therapeutics 11 (12): 2693–703. doi:10.1158/1535-7163.MCT-12-0441-T. PMID 23012245.
  18. ↑ DeRose YS, Wang G, Lin YC, et al. (2011). "Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes". Nature Medicine 17 (11): 1514–20. doi:10.1038/nm.2454. PMC 3553601. PMID 22019887.
  19. ↑ Guenot D, Guérin E, Aguillon-Romain S, et al. (April 2006). "Primary tumour genetic alterations and intra-tumoral heterogeneity are maintained in xenografts of human colon cancers showing chromosome instability". The Journal of Pathology 208 (5): 643–52. doi:10.1002/path.1936. PMID 16450341.
  20. ↑ Uronis JM, Osada T, McCall S, et al. (2012). "Histological and molecular evaluation of patient-derived colorectal cancer explants". PLOS ONE 7 (6): e38422. doi:10.1371/journal.pone.0038422. PMC 3366969. PMID 22675560.
  21. ↑ Krumbach R, Schüler J, Hofmann M, Giesemann T, Fiebig HH, Beckers T (May 2011). "Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance". European Journal of Cancer 47 (8): 1231–43. doi:10.1016/j.ejca.2010.12.019. PMID 21273060.
  22. ↑ Bertotti A, Migliardi G, Galimi F, et al. (November 2011). "A molecularly annotated platform of patient-derived xenografts ('xenopatients') identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer". Cancer Discovery 1 (6): 508–23. doi:10.1158/2159-8290.CD-11-0109. PMID 22586653.
  23. ↑ Hidalgo M, Bruckheimer E, Rajeshkumar NV, et al. (August 2011). "A pilot clinical study of treatment guided by personalized tumorgrafts in patients with advanced cancer". Molecular Cancer Therapeutics 10 (8): 1311–6. doi:10.1158/1535-7163.MCT-11-0233. PMID 21673092.
  24. ↑ Laheru D, Shah P, Rajeshkumar NV, et al. (December 2012). "Integrated preclinical and clinical development of S-trans, trans-Farnesylthiosalicylic Acid (FTS, Salirasib) in pancreatic cancer". Investigational New Drugs (Springer Science+Business Media) 30 (6): 2391–9. doi:10.1007/s10637-012-9818-6. PMC 3557459. PMID 22547163.
  25. 1 2 Braekeveldt N, Wigerup C, Gisselsson D, et al. (March 2015). "Neuroblastoma patient-derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours". International Journal of Cancer 136 (5): E252–61. doi:10.1002/ijc.29217. PMC 4299502. PMID 25220031.
  26. 1 2 "Pediatric Preclinical Testing Program (PPTP)." Pediatric Preclinical Testing Program. Nationwide Children's Hospital, 2009. http://pptp.nchresearch.org
  27. ↑ "Pediatric Preclinical Testing Program (PPTP)". CTEP. August 4, 2014. Retrieved August 31, 2014.
  28. ↑ Houghton PJ, Morton CL, Tucker C, et al. (December 2007). "The pediatric preclinical testing program: description of models and early testing results". Pediatric Blood & Cancer (John Wiley & Sons) 49 (7): 928–40. doi:10.1002/pbc.21078. PMID 17066459.
  29. ↑ Pollack, Andrew (September 25, 2012). "Seeking cures, patients enlist ice stand-ins". Business Day. The New York Times.
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