resection of the primary tumor from mice

Avaliação de células T CD8+ específicas do antígeno durante metástase linfonodal

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.

Autor em destaque: Um modelo para estudar a dinâmica sistêmica e local de células T CD8 + durante a metástase LN

Modelo de Metástase de Linfonodo Drenante para Avaliação da Dinâmica de Células T CD8+ Antígeno-Específicas Durante a Tumorigênese

Ressecção do tumor primário de camundongos

Para começar, palpar o tumor primário e determinar se o tumor tem aproximadamente cinco milímetros de diâmetro. Para realizar a ressecção do tumor, coloque o camundongo em uma cabine de biossegurança e posicione-o sobre uma placa anatômica coberta com papel absorvente limpo. Orientar o camundongo em decúbito dorsal para que seu eixo longitudinal fique paralelo ao experimentador.Desinfete o abdômen do camundongo usando uma bola de algodão embebida em iodopovidona. Em seguida, usando um bisturi estéril, faça uma incisão na pele perto do local portador do tumor. Insira a ponta fechada da pinça estéril na incisão para expor claramente o tumor.Remova o tumor, garantindo que a cápsula permaneça o mais intacta possível. Remova cuidadosa e suavemente o tecido conjuntivo adjacente ao tumor usando tesoura estéril.

Research

JoVE Journal

Immunology and Infection

Resection of the Primary Tumor From Mice

228 visualizações. Para começar, palpar o tumor primário e determinar se o tumor tem aproximadamente cinco milímetros de diâmetro. Para realizar a ressecção do tumor, coloque o camundongo em uma cabine de biossegurança e posicione-o sobre uma placa anatômica coberta com papel absorvente limpo. Orientar o camundongo em decúbito dorsal para que seu eixo longitudinal fique paralelo ao experimentador.Desinfete o abdômen do camundongo usando uma bola de algodão embebida em iodopovidona. Em seguida,...

Vídeo: Ressecção do tumor primário de camundongos

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. ...