Application of Hu-PDX Model in Tumor Immunology

The patient-derived tumor xenograft (PDX) model derived from the patient's primary tumor tissue differs from the traditional cell-derived xenograft (CDX) model and genetically engineered mouse model, which is directly established by using the patient's tumor tissue. The subcutaneous xenograft tumor model can better retain the special functional gene structure and biological markers of the tumor in the individual, which is closer to the individual clinical biological characteristics and can reproduce the complex state of human cancer, such as the heterogeneity of tumor cells. It is an internationally recognized mouse xenograft tumor model that best represents the genetic information characteristics of human tumors and can provide better predictions for drug screening and efficacy observation. In recent years, PDX technology has developed rapidly, showing its essential role in drug screening, curative effect evaluation, prediction of clinical treatment effects during the same period, and analysis of the cause of drug resistance in the late clinical stage. Under certain conditions, the prediction accuracy of PDX samples for the effective rate and drug resistance rate of traditional chemotherapy drugs can reach 90% and 97%. In recent years, humanized immune PDX models have been established based on PDX models so that they can Comprising human tumor cells and immune cells; this model is a translational medicine platform with great potential for evaluating the effects of novel immunotherapeutic agents. ^[1]

The human tumor transplantation model PDX retains the characteristics of the patient's tumor tissue and is more clinically relevant. It can establish an ideal tumor immunotherapy evaluation model combined with humanized mice. Medicilon has developed a variety of PDX models for colon cancer, lung cancer, breast cancer, gastric cancer, etc. At the same time, we have humanized tumor mouse models of the recombinant human immune system, including PBMC humanized tumor models and CD34+ HSC humanized tumor models for customers to choose.

What are the disadvantages of the PDX model, and what is the hu-pdx model?

The tumor xenograft model (patient-derived xenograft, PDX) established by transplanting the patient's tumor tissue into immunodeficient mice well preserved the primary tumor's biological characteristics, but the tumor tissue's matrix components would be lost in the passage of mice. The tumor microenvironment will be changed in mice lacking the immune system, and the heterogeneity of the primary tumor will be gradually lost, which limits the application of this type of model; human hematopoietic stem cells (hematopoietic stem cells, HSCs) were transplanted into severely immunodeficient mice whose bone marrow was cleared by low-dose X-ray whole-body irradiation or other methods, and then the patient's tumor tissue was transplanted into the humanized tumor model (humanized patient-derived xenograft, Hu-PDX), which has a human-like immune system and can better preserve the heterogeneity of the primary tumor. Studies have confirmed that human HSCs can produce various immune cells in mice, making the microenvironment of tumor growth more similar to that of the human body, better retaining the heterogeneity of tumors, and providing a suitable animal model for individualized research on tumors.

Establishment of humanized patient-derived xenograft (Hu-PDX)

Human hematopoietic stem cells（HSCs) were transplanted into X-ray-irradiated severe immunodeficiency mice, and then the patient's tumor tissue was transplanted into the mice to establish a humanized tumor model (humanized patient-derived xenograft, Hu- PDX) can provide tumors with a growth environment more similar to that of the human body. At the same time, the tumor stroma contains human immune cells and cytokines, and other components, which have important applications in the study of targeted tumor therapy and tumor occurrence, development, and metastasis mechanisms. Value, especially in the study of tumor immunotherapy, is a more ideal tumor model.

The establishment method of the Hu-PDX model

The establishment of Hu-PDX model is divided into two parts:

(1) Humanized immune system mice (HISM). There are three commonly used immune system reconstruction methods: one is to co-transplant human bone marrow, liver, and thymus tissues into mice (bonemarrow-liver-thymus, BLT). Although this model has a better immune reconstruction effect, the immune reconstruction materials The source is limited, and the trauma to the patient is great, so it is not suitable for the research of individualized treatment of clinical patients;

Another method is to transplant human peripheral blood mononuclear cells (PBMCs) into mice. This method is simple to operate and can obtain a large number of cells, but the human leukocyte antigen produced by the transplanted PBMCs (human leukocyte antigen, HLA) restricts the production of T cells and NK cells in mice, and mice will develop severe graft-versus-host disease (graft-versus-hostdisease, GVHD) about four weeks after transplantation, thus limiting time to experiment with the model;

Transplantation of human blood CD34+ HSCs (isolated from PBMCs using immunomagnetic beads) into immunodeficient mice is currently the most commonly used method for preparing Hu-PDX models. The effect of bone marrow transplantation can also be evaluated by detecting the expression of CD34+. Amplify the markers of HSCs, which have strong proliferative ability.

(2) The patient's tumor tissue was transplanted into HISM mice to establish the Hu-PDX model. Usually, four weeks after immunodeficiency mice are inoculated with human immune cells (HISM), the patient's tumor tissue is transplanted into the body, the tumor can grow, and the human-derived immune cell components and cytokines produced in the mouse interact with the tumor. Human immunotherapy targets and cytokines can be detected in the model tumor tissue. The tumor shows better tumor heterogeneity in terms of pathological morphology and gene integrity, marking the successful establishment of the Hu-PDX model.

Although hematopoietic stem cells are not easy to obtain in large quantities, which limits their development. However, with the maturity of hematopoietic stem cell culture technology, hematopoietic stem cells can be expanded several times in-vitro under the stimulation of cytokines combined with a special medium. But in addition, it takes at least one month to rebuild the immune system of mice, and it usually takes 2 to 4 months for fresh tumor tissue to be successfully transplanted in mice. The whole process takes a long time and costs a lot. ^[2]

Hu-PDX model construction

The PDX (patient-derived xenograft) model established based on clinical tumor specimens can better maintain the characteristics of the primary tumor, but due to the lack of the human immune system, it is impossible to carry out immunotherapy research on tumor cells or tissues of specific patients. The model established by transplanting the tumor tissue of a specific patient into the humanized immune system mouse is called Hu-PDX (humanized patient-derived xenograft) model because it can simulate the interaction between tumor cells and the immune system in the human body, has an important application prospect in the research of anti-tumor immunotherapy. The construction of the Hu-PDX model is closely related to the time of tumor transplantation and the method of humanization. For example, the immune reconstitution of the Hu-PBL mouse model lasts for a short time, so the construction of the Hu-PBL-PDX model is usually transplanted with the tumor tissue of the patient first, and the mice are sublethal when the tumor volume reaches about 120-180 mm3. After sexual irradiation, human PBMCs were injected through the tail vein; the Hu-HSC mouse model has a weak GVHD response, and the immune reconstitution lasts for about 10-12 weeks, so the Hu-HSC-PDX model is usually injected with human CD34 + HSCs were transplanted into immunodeficient mice treated with sublethal irradiation, and when the human CD3 + CD45 + ratio in the mice exceeded 15% (usually 12 weeks after transplantation), they were then transplanted into tumor tissues of patients. The sign of the successful construction of this model is that human immune cells and cytokines can be detected in tumor tissue. ^[3]

Application of humanized mouse model in tumor immunotherapy research

Tumor immunotherapy is to control and kill tumors by activating the body's anti-tumor immune response or blocking tumor immune escape. It mainly includes chimeric antigen receptor T cells (chimeric antigen receptor T cells, CAR-T), immune checkpoint inhibitor therapy, and comprehensive therapy combined with other immunotherapies. Ideal preclinical model for therapeutic research.

Application of CAR-T therapy

The principle of CAR-T cell immunotherapy is that T cells modified by chimeric antigen receptors can specifically recognize tumor-associated antigens, so that the targeting and killing activity of effector T cells are higher than those of conventional immune cells, thus exerting anti-inflammatory effects. cancer effect. At present, CAR-T therapy is mainly used clinically in the treatment of hematological malignancies such as B-cell lymphoma and leukemia, but is rarely used in the treatment of solid tumors. Humanized mouse models have been applied to evaluate the anti-tumor efficacy of various CAR designs. Abate-Daga et al. developed a CAR targeting prostate stem cell antigen (PSCA) using the pancreatic cancer Hu-PBMC-PDX model, providing evidence for using PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer; another study reported It was proved that CD27 can co-stimulate CAR-T cells to obtain higher persistence and anti-tumor activity in the Hu-PBMC-PDX model of ovarian cancer. Obviously, the Hu-PDX model provides a new method for evaluating the effectiveness of CAR-T therapy in solid tumors.

Medicilon's PDX Model

Now, Medicilon has the PDX models covering colon cancer, lung cancer, gastric cancer, breast cancer, liver cancer, and pancreas cancer. Our research on the PDX model includes molecular-level genotyping and pharmacological efficacy evaluation service of the orthotopic model, promising great predictions for clinical efficacy research.

Reference:

[1]. ZHOU Zhen, XU Yun-hua, et al. Establishment of a lung cancer mouse model with humanized immunity and its role in efficacy evaluation of programmed death-1 inhibitors. Journal of Shanghai Jiaotong University.

 [2]. Ningning Zhao, et al. Research progress on humanized patient-derived xenograft (Hu-PDX) models. Acta Laboratorium Animalis Scientia Sinica. 2018,26(06):804-808.

[3]. Guo WW, Qiao TY, Zhang CQ, et al. Establishment of mouse models with a humanized immune system and applications for tumor immunotherapy [J]. Chin J Comp Med, 2019, 29(11): 98-104. doi: 10. 3969 / j.issn.1671-7856. 2019. 11. 015