ectopic inoculation of b16f10-gp melanoma cells in inguinal region of mice

评估淋巴结转移过程中抗原特异性 CD8+ T 细胞

Cancer immunotherapy, especially the immune checkpoint blockade (ICB), has revolutionized cancer therapy1. ICB blocks the coinhibitory immunoreceptors (such as PD-1, Tim-3, LAG-3, and TIGIT), which are highly expressed in exhausted CD8+ T cells in the tumor microenvironment (TME), leading to the reinvigoration of exhausted CD8+ T cells2. Considering the heterogeneity of exhausted CD8+ T cells, accumulating evidence revealed that tumor-specific CD8+ T cells derived from the periphery, including draining lymph node (dLN), but not in TME, mediate the efficacy of ICB3,4,5,6,7,8. Recently, TdLN derived TCF-1+TOX- tumor-specific memory CD8+ T cells (TdLN-TTSM) was confirmed to be the genuine responders to ICB which embody several functional properties of conventional memory T cells and could further expand and differentiate into progeny exhausted cells upon ICB treatment9. Altogether, these findings corroborated the importance of LN in mounting anti-tumor immunity.
Lymph node functions as a critical place in facilitating the priming and activation of tumor-specific CD8+ T cells by providing structural basis as well as biological signals10. Several types of cancer cells frequently seed sentinel lymph node (SLN, the first LN draining a primary tumor) before systematic dissemination11. The presence of SLN metastasis is linked with poor outcome in human cancer and preclinical models showed that tumor cells in TdLN could spread to distant organs through both the lymphatic vessels and blood vessels of the node12,13,14,15. SLN biopsy now represents a standard procedure to guide subsequent treatment decisions in many solid tumor types which could avoid unnecessary resection of uninvolved LN16,17. Even to the involved LN, it remains controversial whether and when surgical resection is needed as several studies have demonstrated that the removal of regional LN did not exhibit improved overall survival compared to those that received radiation or systemic therapy without regional LN resection18,19. One interpretation is that metastatic LN (mLN) with microscopic disease may retain some capacity to educate immune cells and provide some therapeutic benefits. So, it is critically important to elucidate how LN metastasis affects the anti-tumor immune response, especially the properties and functions of TdLN-TTSM.
Until now, both preclinical and clinical data have revealed some structural and cellular alterations in mLN20. However, the dynamic changes of tumor-specific CD8+ T cells during LN metastasis have not been delineated. Therefore, developing a compelling model of LN metastasis is needed for further investigation. Indeed, several studies have reported mLN mouse models through different ways14,21,22. For example, spontaneous metastasis in axillary LNs was conducted through the implantation of 4T1 breast cancer cells into the mammary fat pad22. In another study, Reticker-Flynn et al. generated melanoma cell lines with high incidence of spread from subcutaneous primary tumor to LNs through serial inoculation of tumor cells cultured from dissociated mLN tissues (nine rounds)14. Another commonly used model was prepared by the injection of tumor cells into the footpad and the metastatic loci would be formed in popliteal LN22. Notably, it is difficult to evaluate the precise timepoints of intervention because LN metastasis in these models is not always faithful.
In the present study, a murine LN metastatic model was established through the intranodal injection of B16F10-GP cells23,24, generated by CRISPR/Cas9-mediated insertion of LCMV virus glycoprotein (GP) gene sequence into the genome of B16F10 cell line9. Then, these mice were transferred with P14 cells which harbor transgenic T cell receptors (TCRs) specifically recognize the H-2Db GP33-41 epitope25,26 and the systemic and local dynamics of antigen-specific CD8+ T cells during LN metastasis could be investigated. Our experimental design provides a useful model for the study of immune responses, especially the antigen-specific CD8+ T cells during the LN metastasis which excludes the perturbation of bystander CD8+ T cells. These results would affect the clinical treatment options of whether to remove or retain the mLN and shed new light on the manipulation of mLN to achieve maximum therapeutic benefits.

作者聚焦：研究 LN 转移期间 CD8+ T 细胞全身和局部动力学的模型

用于评估肿瘤发生过程中抗原特异性 CD8+ T 细胞动力学的引流淋巴结转移模型

This protocol provides a useful reproductive model for the studies of antigen specific CD8 T-cells. They are lymph node metastasis, which excludes the participation of bystander CD8 T-cells. We try to reveal the systemic and local dynamics of antigen specific CD8 T cells They are lymph node metastasis.Recent accumulating evidence revealed that tumor specific CD8 T cells derived from the periphery, especially in tumor draining lymph node, but not in tumor microenvironment met the efficacy of immune checkpoint blockage therapy. Recently we identified this in one positive talks negative tumor sub specific memory T cells in tumor draining lymph nodes of both mice tumor models and human hepatocellular carcinoma patients which serve as general responders to immune checkpoint blockage. It is difficult to evaluate the precise time points of intubation because lymph nodes metastasis through other techniques is not always feasible.This protocol has provided an approach to precisely investigate the antigen specific CD8 T-cells immune responses. The lymph node metastasis, which excludes the participation of bystander CD8 T cells. Our experimental design provides a useful model to investigate the antigen specific CD8 T-cells, the lymph node metastasis and interactions between antigen specific CD8 T cells and other immune cells or stroma cells during lymph node metastasis.We'll try to elucidate how lymph node metastasis affects the anti-tumor immune response, especially the properties and the functions of TDR and TTSM cells. This results would affect the clinical treatment options of whether to remove or retain the MLN and show the new light on the manipulation of MLN to achieve maximum therapeutic benefits.

小鼠腹股沟区B16F10-GP黑色素瘤细胞的异位接种

首先，准备 B16 F10 GP 黑色素瘤细胞悬液。在吸出细胞悬液之前，轻弹管壁以将气泡移动到顶部。使用一毫升注射器吸出 100 微升细胞悬浮液，并推动活塞以将顶部气泡移出。接下来，将鼠标置于仰卧位，露出腹部。用手指按压鼠标的左后肢，使左腹股沟区域的皮肤完全暴露。剃掉小鼠的左下腹部，并用含有75%乙醇的棉花清洁。然后将针头插入角度保持在大约45度，向上倾斜，将针头插入左大腿上腹股沟区域的皮下组织中，深度为0.5至1厘米。轻轻插入针头。同时，将细胞缓慢注射到皮下组织中。观察一个小的推注，表示在皮下区域形成一个液体袋。注射后，扣上保护套，将其丢弃在锐器盒中，然后将鼠标放回笼子中。

ectopic-inoculation-b16f10-gp-melanoma-cells-inguinal-region

Research

JoVE Journal

Immunology and Infection

Ectopic Inoculation of B16F10-GP Melanoma Cells in Inguinal Region of Mice

156 次观看.  Qingdao Agricultural University. 首先，准备 B16 F10 GP 黑色素瘤细胞悬液。在吸出细胞悬液之前，轻弹管壁以将气泡移动到顶部。使用一毫升注射器吸出 100 微升细胞悬浮液，并推动活塞以将顶部气泡移出。接下来，将鼠标置于仰卧位，露出腹部。用手指按压鼠标的左后肢，使左腹股沟区域的皮肤完全暴露。剃掉小鼠的左下腹部，并用含有75%乙醇的棉花清洁。然后将针头插入角度保持在大约45度，?...

视频: 小鼠腹股沟区B16F10-GP黑色素瘤细胞的异位接种

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. ...