Patient derived xenograft

Several types of immunodeficient mice can be used to establish PDX models: athymic nude mice, severely compromised immune deficient (SCID) mice, NOD-SCID mice, and recombination-activating gene 2 (Rag2)-knockout mice. 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 PDX models because the NOD-SCID mouse does not produce natural killer cells. When human tumors are resected, necrotic tissues are removed and the tumor can be mechanically sectioned into smaller fragments, chemically digested, or physically manipulated into a single-cell suspension. There are 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, a 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. Heterotopic and orthotopic implantation Unlike creating xenograft mouse models using existing cancer cell lines, there are no intermediate in vitro processing steps before implanting tumor fragments into a murine host to create a PDX. The tumor fragments are either implanted heterotopically or orthotopically into an immunodeficient mouse. With heterotopic implantation, the tissue or cells are implanted into an area of the mouse unrelated to the original tumor site, generally subcutaneously or in subrenal capsular sites. The advantages of this method are the direct access for implantation, and ease of monitoring the tumor growth. With orthotopic implantation, scientists transplant the patient's tumor tissue or cells into the corresponding anatomical position in the mouse. Subcutaneous PDX models rarely produce metastasis in mice, nor do they simulate the initial tumor microenvironment, with engraftment rates of 40-60%. Ultimately, 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. Generations of engraftments 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 PDX has not genetically or histologically diverged from the patient's tumor. ==Advantages over established cancer cell lines==

Cancer cell lines are originally derived from patient tumors, but acquire the ability to proliferate within in vitro cell cultures. As a result of in vitro manipulation, cell lines that have been traditionally used in cancer research undergo genetic transformations that are not restored when cells are allowed to grow in vivo. 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. When implanted into immunodeficient mice, cell lines do not easily develop tumors and the result of any successfully grown tumor is a genetically divergent tumor unlike the heterogeneous patient tumor. Specifically, cell line-xenografts often are not predictive of the drug response in the primary tumors because cell lines do not follow pathways of drug resistance or the effects of the microenvironment on drug response found in human primary tumors. Since PDX can be passaged without in vitro processing steps, PDX models allow the propagation and expansion of patient tumors without significant genetic transformation of tumor cells over multiple murine generations. Within PDX models, patient tumor samples grow in physiologically-relevant tumor microenvironments that mimic the oxygen, nutrient, and hormone levels that are found in the patient's primary tumor site. As a result, numerous studies have found that PDX models exhibit similar responses to anti-cancer agents as seen in the actual patient who provided the tumor sample. Humanized xenograft models One prominent shortcoming of PDX models is that immunodeficient mice must be used to prevent immune attacks against the xenotransplanted tumor. With the immune system incapacitated, a critical component of the known tumor microenvironment interaction is foregone, preventing immunotherapies and anti-cancer agents that target the immune system components from being studied in PDX models. Researchers are beginning to explore the use of humanized-xenograft models to enable immune studies. Humanized-xenograft models are created by co-engrafting the patient tumor fragment and peripheral blood or bone marrow cells into a NOD/SCID mouse. However, these strategies have yet to be validated for most tumor types and there remain 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. Humanized-xenograft models for acute lymphoblastic leukemia and acute myeloid leukemia have been created. ==Clinical relevance==

Breast cancer The classification of genetic breast cancer subtypes, including triple-negative and HER2-positive subtypes, Scientists have also found that breast cancer PDX 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. Breast cancer brain metastases affect younger women disproportionally, especially those lacking estrogen-receptor (ER), progesterone-receptor, and HER2 (known as triple-negative breast cancer, TNBC). Contreras-Zarate MJ et al. developed and characterized novel heterogeneous and clinically relevant human brain metastasis breast cancer PDXs (BM-PDXs) to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings. Colorectal cancer Colorectal PDX models are relatively easy to establish and the models maintain genetic similarity of primary patient tumor for about 14 generations. In 2012, a study established 27 colorectal PDX models that did not diverge from their respective human tumors in histology, gene expression, or KRAS/BRAF mutation status. Due to their stability, the 27 colorectal PDX models may be able to serve as pre-clinical models in future drug studies. Drug resistance studies have been conducted using colorectal PDX models. In one study, researchers found that the models predicted patient responsiveness to cetuximab with 90% accuracy. Another study identified the amplification of ERBB2 as another mechanism of resistance, and a putative new actionable target in treatments. Pancreatic cancer Researchers initially focused on using pancreatic PDX models for drug studies to improve the process to develop predictive and pharmacodynamics end points for several molecularly targeted therapies. Pancreatic PDX models have shown anti-mesothilin CAR-T cells (T-cells modified with a chimeric antigen receptor) to suppress cancer growth. Pediatric cancer (neuroblastoma) Researchers have established neuroblastoma PDXs by orthotopic implantation of patient tumor explants into immunodeficient mice. The PDXs retained the genotype and phenotype of patient tumors, and exhibited substantial infiltrative growth and metastasis to distant organs including the bone marrow. The researchers cultured PDX-derived neuroblastoma cells in vitro and the cells retained tumorigenic and metastatic capacity in vivo. Brain cancer (Glioblastoma) PDX models of glioblastoma (GBM) have been essential for improving our understanding of the disease both in preclinical and translational research. In vitro cell culture models of glioblastoma, although valuable, can not fully replicate the complexity of the disease since there is a clear lack of the brain microenvironment and clonal selection. Orthotopic PDXs of GBM can be established through intracranial injections of tumor cells using a stereotactic frame. It has been shown that PDX models of GBM can recapitulate the histopathology, phenotypic properties and genetics of the parental patient tumor, highlighting the relevance of such models for GBM research. ==Challenges with PDX model adaptation ==

There are several challenges that scientists face when developing or using PDX models in research. For instance, not all tumor samples will successfully engraft in an immunodeficient mouse. When engraftment does occur, clinical study protocols are difficult to standardize if engraftment rates vary. Further, it is expensive to house mice, maintain histopathological cores for frequent testing, and USA, and the Horizon 2020 program is funding a new Research Infrastructure providing standardised services and resources, with the goal of improving reproducibility and open access to resources and services. With regard to using PDX in personalized medicine, there are financial challenges. In the US, the cost to develop PDX models can potentially cost a patient thousands of dollars for treatment. PDX models can also take significant time to create, which may pose a challenge to patients with advanced stages of cancer. == References ==