CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor.

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ T cells. Murine tumor models revealed that tumor-specific CD8+ T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ T cell activation in response to cancer is different from that of canonical CD8+ T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

Prognostic effect of TCF1+ CD8+ T cell and TOX+ CD8+ T cell infiltration in lung adenocarcinoma.

Recent studies have highlighted the pivotal roles of T cell transcription factors TCF-1 and TOX in modulating the immune response in cancer, with TCF-1 maintaining CD8+ T cell stemness and TOX promoting T cell exhaustion. The prognostic significance of these factors in lung adenocarcinoma (LUAD) remains a critical area of investigation. The retrospective study included 191 patients with LUAD who underwent surgery, of whom 83% were in stages II and III. These patients were divided into exploratory (n = 135) and validation (n = 56) groups based on the time of diagnosis. Multiplex fluorescence immunohistochemistry was used to examine the infiltration levels of CD8+ T cells, TCF1+ CD8+ T cells, and TOX+ CD8+ T cells. The percentage of CD8+ T cells in tumor was markedly lower than that in stroma (p < 0.05). In tumor-draining lymph nodes (TDLNs) invaded by tumor, the proportion of stem-like TCF1+ CD8+ T cells was significantly decreased (p < 0.01). Importantly, higher infiltration levels of CD8+ T cells and TCF1+ CD8+ T cells were associated with improved disease-free survival (DFS) (p = 0.009 and p = 0.006, respectively) and overall survival (OS) (p = 0.018 and p = 0.010, respectively). This study underscores the potential of TCF1+ CD8+ T cells as prognostic biomarkers in LUAD, providing insights into the tumor immune microenvironment and guiding future therapeutic strategies.

Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes.

The contribution of different immune cell subsets, especially T cells, in anti-tumor immune response is well established. In contrast to T cells, the anti-tumor contribution of B cells has been scarcely investigated. B-cells are often overlooked, even though they are important players in a fully integrated immune response and constitute a substantial fraction of tumor draining lymph nodes (TDLNs) known also as Sentinel Nodes. In this project, samples including TDLNs, non-TDLNs (nTDLNs) and metastatic lymph nodes from 21 patients with oral squamous cell carcinoma were analyzed by flow cytometry. TDLNs were characterized by a significantly higher proportion of B cells compared with nTDLNs (P = .0127). TDLNs-associated B cells contained high percentages of naïve B cells, in contrary to nTDLNs which contained significantly higher percentages of memory B cells. Patients having metastases in TDLNs showed a significantly higher presence of immunosuppressive B regulatory cells compared with metastasis-free patients (P = .0008). Elevated levels of regulatory B cells in TDLNs were associated with the advancement of the disease. B cells in TDLNs were characterized by significantly higher expression of an immunosuppressive cytokine-IL-10 compared with nTDLNs (P = .0077). Our data indicate that B cells in human TDLNs differ from B cells in nTDLNs and exhibit more naïve and immunosuppressive phenotypes. We identified a high accumulation of regulatory B cells within TDLNs which may be a potential obstacle in achieving response to novel cancer immunotherapies (ICIs) in head and neck cancer.

Automated detection of vascular remodeling in tumor-draining lymph nodes by the deep-learning tool HEV-finder.

Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Regulatory and effector T cell subsets in tumor-draining lymph nodes of patients with squamous cell carcinoma of head and neck.

BACKGROUND: A crucial role for the immune system has been proposed in the establishment and progression of head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the cytokine and regulatory profiles of T cells in tumor draining lymph nodes (TDLNs) of patients with HNSCC. RESULTS: The frequencies of CD4+TNF-α+ and CD4+TNF-αhi negatively were associated with poor prognostic factors such as LN involvement (P = 0.015 and P = 0.019, respectively), stage of the disease (P = 0.032 and P = 0.010, respectively) and tumor size (P = 0.026 and P = 0.032, respectively). Frequencies of CD8+IFN-γ+ and CD8+IFN-γ+ TNF-α+ T cells showed negative relationship with tumor grade (P = 0.035 and P = 0.043, respectively). While, the frequencies of CD4+IL-4+, CD8+IL-10+, CD8+IL-4+T cells were higher in advanced stages of the disease (P = 0.042, P = 0.041 and P = 0.030, respectively) and CD4+IFN-γ+TNF-α-, CD8+IL-4+ and CD8+IFN-γ+TNF-α- T cells were higher in patients with larger tumor size (P = 0.026 and P = 0.032, respectively). Negative associations were found between the frequencies of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3+CD127low/- Treg cells and cancer stage (P = 0.015 and P = 0.059). CONCLUSION: This study shed more lights on the changes in immune profile of T cells in TDLNs of HNSCC. Larger tumor size and/or LN involvement were associated with lower frequencies of CD4+TNF-α+, CD8+IFN-γ+ and CD8+IFN-γ+TNF-α+ but higher frequency of CD4+IL-4+ T cells. Moreover, Foxp3+Tregs correlated with good prognostic indicators.

Targeting tumor extracellular matrix activates the tumor-draining lymph nodes.

Disruption of the tumor extracellular matrix (ECM) may alter immune cell infiltration into the tumor and antitumor T cell priming in the tumor-draining lymph nodes (tdLNs). Here, we explore how intratumoral enzyme treatment (ET) of B16 melanoma tumors with ECM-depleting enzyme hyaluronidase alters adaptive and innate immune populations, including T cells, DCs, and macrophages, in the tumors and tdLNs. ET increased CD103+ DC abundance in the tdLNs, as well as antigen presentation of a model tumor antigen ovalbumin (OVA), eliciting local OVA-specific CD8+ T cell responses. Delivered in combination with a distant cryogel-based cancer vaccine, ET increased the systemic antigen-specific CD8+ T cell response. By enhancing activity within the tdLN, ET may broadly support immunotherapies in generating tumor-specific immunity.

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy.

BACKGROUND: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy. METHODS: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs). RESULTS: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells. CONCLUSION: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT.

Correlation and prognostic implications of intratumor and tumor draining lymph node Foxp3+ T regulatory cells in colorectal cancer.

BACKGROUND: The prognostic value of intratumor T regulatory cells (Tregs) in colorectal cancer (CRC) was previously reported, but the role of these cells in tumor draining lymph nodes (TDLNs) was less addressed. METHODS: A total of 150 CRC stages I-IV were retrospectively enrolled. Intratumor and TDLN Tregs were examined by immunohistochemical assay. The association of these cells was estimated by Pearson correlation. Survival analyses of subgroups were conducted by Kaplan-Meier curves, and the log-rank test and risk factors for survival were tested by the Cox proportional hazard model. RESULTS: High accumulation of Tregs in tumors was significant in patients with younger age and good histological grade, where enrichment of these cells in TDLNs was more apparent in those with node-negative disease and early TNM stage disease, both of which were more common in early T stage cases. A significant correlation of intratumoral and TDLN Tregs was detected. Patients with higher intratumoral Tregs displayed significantly better PFS and OS than those with lower Tregs. However, no such differences were found, but a similar prognostic prediction trend was found for these cells in TDLNs. Finally, intratumoral Tregs were an independent prognostic factor for both PFS (HR = 0.97, 95% CI 0.95-0.99, P < 0.01) and OS (HR = 0.98, 95% CI 0.95-1.00, P = 0.04) in the patients. CONCLUSIONS: Higher intratumor Tregs were associated with better survival in CRC. Although no such role was found for these cells in TDLNs, the positive correlation and similar prognostic prediction trend with their intratumoral counterparts may indicate a parallelized function of these cells in CRC.

CD4+ and CD8+ T cells in sentinel nodes exhibit distinct pattern of PD-1, CD69, and HLA-DR expression compared to tumor tissue in oral squamous cell carcinoma.

Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti-programmed cell death protein 1 (PD-1) treatment is predominantly studied in tumor-infiltrating lymphocytes (TILs). Its impact on PD-1 expressing cells in tumor-draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD-1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD-1, CD69, and HLA-DR on CD4+ and CD8+ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD-1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD-1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD-1 negative TILs expressed high levels of human leukocyte antigen - DR isotype and CD69. In contrast, PD-1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3+ PD-1+ cells in tumor-draining lymph nodes had significantly lower disease-free and overall survival rates (log-rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD-1 levels in TDLNs correspond with survival and potentially with response to anti-PD-1 therapy. Such knowledge may ultimately help guide anti-PD-1 treatment.

Immune Checkpoint Therapy: Tumor Draining Lymph Nodes in the Spotlights.

Tumor-draining lymph nodes play a paradoxical role in cancer. Surgeons often resect these sentinel lymph nodes to determine metastatic spread, thereby enabling prognosis and treatment. However, lymph nodes are vital organs for the orchestration of immune responses, due to the close encounters of dedicated immune cells. In view of the success of immunotherapy, the removal of tumor-draining lymph nodes needs to be re-evaluated and viewed in a different light. Recently, an important role for tumor-draining lymph nodes has been proposed in the immunotherapy of cancer. This new insight can change the use of immune checkpoint therapy, particularly with respect to the use in neoadjuvant settings in which lymph nodes are still operational.

Detection of micro-metastases by flow cytometry in lymph nodes from patients with penile cancer.

BACKGROUND: The tumor draining lymph node concept was first described in penile cancer for staging. Immunohistochemistry and histopathology evaluations are routinely used in clinical practice to examine lymph nodes for metastasis. However, these methods are time-consuming with low diagnostic accuracy and micro-metastases might be missed. In this study, we aim to evaluate detection of metastatic cells in draining lymph nodes by flow cytometry. METHODS: To assess the sensitivity of micro-metastasis detection by FACS (Fluorescence-activated cell sorting), HeLa cells were titrated into Peripheral blood mononuclear cells (PBMCs) and expression of pan-cytokeratin AE1/AE3 was analyzed. Single cell suspensions were separately prepared from 10 regional lymph nodes obtained from 5 patients with invasive penile cancer undergoing radical surgery and lymph node dissection. Lymph node dereived cells were examined for cell surface expression of EpCAM, E-cadherin and intracellular expression of pan-cytokeratin AE1/AE3 by FACS. RESULTS: Ten lymph nodes from 5 penile cancer patients were investigated in a head-to-head comparison between FACS and pathology examination of sections. All metastatic lymph nodes verified by pathology examination were also identified by FACS. Two additional lymph nodes with micro-metastases were diagnosed by FACS only. CONCLUSIONS: FACS analyses of pan-cytokeratin AE1/AE3 stained single cells from tumor draining lymph nodes can be used to detect micro-metastases in patients with penile cancer patients.

Reduced number of CD169+ macrophages in pre-metastatic regional lymph nodes is associated with subsequent metastatic disease in an animal model and with poor outcome in prostate cancer patients.

BACKGROUND: Tumor-derived antigens are captured by CD169+ (SIGLEC1+ ) sinus macrophages in regional lymph nodes (LNs), and are presented to effector cells inducing an anti-tumor immune response. Reduced CD169 expression in pre-metastatic regional LNs is associated with subsequent metastatic disease and a poor outcome in several tumor types, but if this is the case in prostate cancer has not been explored. METHODS: CD169 expression was measured with immunohistochemistry in metastasis-free regional LNs from 109 prostate cancer patients treated with prostatectomy (January 1996 to April 2002). Possible associations of CD169 expression with PSA-relapse, prostate cancer death, Gleason score, and other clinical data were assessed using Kaplan-Meier survival- and Cox regression analysis. In addition, the Dunning rat prostate tumor model was used to examine CD169 expression in pre-metastatic LNs draining either highly metastatic MatLyLu- or poorly metastatic AT1-tumors. RESULTS: In patients with low CD169 immunostaining in metastasis-free regional LNs, 8 of the 27 patients died from prostate cancer compared with only three of the 82 patients with high immunostaining (P < 0.001). CD169 expression in regional LNs was not associated with PSA-relapse. Rats with highly metastatic tumors had decreased CD169 immunoreactivity in pre-metastatic regional LNs compared with rats with poorly metastatic tumors. CONCLUSION: Low expression of CD169 in metastasis-free regional LNs indicates a reduced anti-tumor immune response. If verified in other studies, CD169 expression in regional LNs could, in combination with other factors, potentially be used as a marker of prostate cancer aggressiveness.

Lymph node targeting strategy using a hydrogel sustained-release system to load effector memory T cells improves the anti-tumor efficacy of anti-PD-1.

Communication between tumors and lymph nodes carries substantial significance for antitumor immunotherapy. Remodeling the immune microenvironment of tumor-draining lymph nodes (TdLN) plays a key role in enhancing the anti-tumor ability of immunotherapy. In this study, we constructed a biomimetic artificial lymph node structure composed of F127 hydrogel loading effector memory T (TEM) cells and PD-1 inhibitors (aPD-1). The biomimetic lymph nodes facilitate the delivery of TEM cells and aPD-1 to the TdLN and the tumor immune microenvironment, thus realizing effective and sustained anti-tumor immunotherapy. Exploiting their unique gel-forming and degradation properties, the cold tumors were speedily transformed into hot tumors via TEM cell supplementation. Meanwhile, the efficacy of aPD-1 was markedly elevated compared with conventional drug delivery methods. Our finding suggested that the development of F127@TEM@aPD-1 holds promising potential as a future novel clinical drug delivery technique. STATEMENT OF SIGNIFICANCE: F127@TEM@aPD-1 show unique advantages in cancer treatment. When injected subcutaneously, F127@TEM@aPD-1 can continuously supplement TEM cells and aPD-1 to tumor draining lymph nodes (TdLN) and the tumor microenvironment, not only improving the efficacy of ICB therapy through slow release, but also exhibiting dual regulatory effects on the tumor and TdLN.

Methods behind oncolytic virus-based DC vaccines in cancer: Toward a multiphase combined treatment strategy for Glioblastoma (GBM) patients.

Glioblastoma (GBM) remains an orphan cancer disease with poor outcome. Novel treatment strategies are needed. Immunotherapy has several modes of action. The addition of active specific immunotherapy with dendritic cell vaccines resulted in improved overall survival of patients. Integration of DC vaccination within the first-line combined treatment became a challenge, and immunogenic cell death immunotherapy during chemotherapy was introduced. We used a retrospective analysis using real world data to evaluate the complex combined treatment, which included individualized multimodal immunotherapy during and after standard of care, and which required adaptations during treatment, and found a further improvement of overall survival. We also discuss the use of real world data as evidence. Novel strategies to move the field of individualized multimodal immunotherapy forward for GBM patients are reviewed.

Adoptive immunotherapy with cells from tumor-draining lymph nodes activated and expanded in vitro.

Tumor-draining lymph nodes (tumor-DLNs) provide a rich source of tumor-reactive lymphocytes which can be used in adoptive immunotherapy (AIT) and that circumvent the need to resect autologous tumor, without the challenges and shortcomings associated with using autologous tumor or anti-CD3 monoclonal antibody. Bryostatin/Ionomycin (Bryo/Io) provide a useful method of activating tumor-DLNs such that they can readily be expanded to sufficient numbers to be used in AIT, and growing the tumor-DLN lymphocytes in the gamma chain cytokines IL-7 plus IL-15 is superior to IL-2 in terms of T cell numbers and phenotype. AIT with these cells induces tumor regression and provides protection against metastases and future tumor challenge. Here, we provide a stepwise protocol to sensitize tumor-DLN cells in donor mice, activate tumor-DLN T cells ex vivo using Bryo/Io, expansion of these cells in gamma chain cytokines and adoptive transfer of the expanded cells back into tumor-bearing hosts. Methods relevant to these experiments, such as injecting tumor cells intravenously and monitoring for pulmonary metastases, tumor volume measurement and resection, and use of luciferase-expressing tumor cells to monitor for metastases following resection, are described in detail. The methods outlined herein can be easily adapted to suit similar experiments across multiple tumor cell lines and syngeneic mouse models.

Micro-to-Nano Oncolytic Microbial System Shifts from Tumor Killing to Tumor Draining Lymph Nodes Remolding for Enhanced Immunotherapy.

Because the tumor-draining lymph nodes (TDLNs) microenvironment is commonly immunosuppressive, oncolytic microbe-induced tumor antigens aren't sufficiently cross-primed tumor specific T cells through antigen-presenting cells (e.g., dendritic cells (DCs)) in TDLNs. Herein, this work develops the micro-to-nano oncolytic microbial therapeutics based on pyranose oxidase (P2 O) overexpressed Escherichia coli (EcP) which are simultaneously encapsulated by PEGylated mannose and low-concentrated photosensitizer nanoparticles (NPs). Following administration, P2 O from this system generates toxic hydrogen peroxide for tumor regression and leads to the release of tumor antigens. The "microscale" EcP is triggered, following exposure to the laser irradiation, to secrete the "nanoscale" bacterial outer membrane vesicles (OMVs). The enhanced TDLNs delivery via OMVs significantly regulates the TDLNs immunomicroenvironment, promoting the maturation of DCs to potentiate tumor antigen-specific T cells immune response. The micro-to-nano oncolytic microbe is leveraged to exert tumor killing and remold TDLNs for initiating potent activation of DCs, providing promising strategies to facilitate microbial cancer vaccination.

NIR-II Ratiometric Fluorescence Probes Enable Precise Determination of the Metastatic Status of Sentinel Lymph Nodes.

Accurately determining the metastatic status of sentinel lymph nodes (SLNs) through noninvasive imaging with high imaging resolution and sensitivity is crucial for cancer therapy. Herein, we report a dual-tracer-based NIR-II ratiometric fluorescence nanoplatform combining targeted and nontargeted moieties to determine the metastatic status of SLNs through the recording of ratio signals. Ratiometric fluorescence imaging revealed approximately 2-fold increases in signals in tumor-draining SLNs compared to inflamed and normal SLNs. Additionally, inflamed SLNs were diagnosed by combining the ratio value with the enlarged size outputted by NIR-II fluorescence imaging. The metastatic status diagnostic results obtained through NIR-II ratiometric fluorescence signals were further confirmed by standard H&E staining, indicating that the ratiometric fluorescence strategy could achieve distant metastases detection. Furthermore, the superior imaging quality of ratiometric probes enables visualization of the detailed change in the lymphatic network accompanying tumor growth. Compared to clinically available and state-of-the-art NIR contrast agents, our dual-tracer-based NIR-II ratiometric fluorescence probes provide significantly improved performance, allowing for the quick assessment of lymphatic function and guiding the removal of tumor-infiltrating SLNs during cancer surgery.

Immune characteristics associated with lymph node metastasis in early-stage NSCLC.

PURPOSE: Tumor metastasis significantly impacts the prognosis of non-small cell lung cancer (NSCLC) patients, with lymph node (LN) metastasis being the most common and early form of spread. With the development of adjuvant immunotherapy, increasing attention has been paid to the tumor-draining lymph nodes（TDLN) in early-stage NSCLC, especially tumor-metastatic lymph nodes, which provides poor prognostic information but has potential benefits in adjuvant treatment. METHODS: We showed the remodeled immune environment in TDLNs through using TCR-seq to analyse 24 primary lung cancer tissues and 134 LNs from 24 lung cancer patients with or without LN metastasis. Additionally, we characterized the spatial profiling of immunocytes and tumor cells in TDLNs and primary tumor sites through using multi-IHC. RESULTS: We found the remodeled immune environment in TDLNs through analyzing primary lung cancer tissues and LNs from NSCLC patients with or without LN metastasis. Considering the intricate communication between tumor and immunocytes, we further subdivided TDLNs, revealing that metastasis-negative LNs from LN-metastatic patients (MNLN) exhibited greater immune activation, exhaustion, and memory in comparison to both metastasis-positive LNs (MPLN) and TDLNs from non-LN-metastatic patients (NMLN). CONCLUSIONS: Our data indicate that LN metastasis facilitated tumor-specific antigen presentation in TDLNs and induces T cell priming, while existing tumor cells generate an immune-suppressive environment in MPLNs through multiple mechanisms. These findings contribute to a comprehensive understanding of the immunological mechanisms through which LN metastasis influences tumor progression and plays a role in immunotherapy for NSCLC patients.

Albumin-Hitchhiking Drug Delivery to Tumor-Draining Lymph Nodes Precisely Boosts Tumor-Specific Immunity through Autophagy Modulation of Immune Cells.

Tumor-draining lymph nodes (TDLNs) are the first sites where tumor components reach and dendritic cells (DCs) present tumor-associated antigens to T cells. DCs rely on autophagy to process tumor antigens into epitope peptides to form epitope-MHC complexes. Selective delivery of autophagy-stimulating drugs to TDLNs may be a precise strategy to boost chemotherapy-induced antitumor immunity. Here, a multistage stimulating strategy is proposed to activate the antitumor immunity cascade by inducing immunogenic death of tumor cells and elevating antigen presentation of DCs in TDLNs. A tumor-microenvironment-responsive "albumin-hitchhiking" micelle is established by self-assembling tumor-targeting oxaliplatin prodrug and lipophilized trehalose prodrug. This demonstrates that lipophilic modification of trehalose with a DSPE tail and the precise exposure in the tumor site enhances its binding to endogenous albumin and realizes TDLNs-selective reflux, where it upregulates antigen processing and presentation of DCs. This study introduces an approach for targeted delivery to TDLNs and provides insights into mechanisms of autophagy in tumor-specific immunity.

Preclinical Identification Of Tumor-Draining Lymph Nodes Using a Multimodal Non-invasive In vivo Imaging Approach.

PURPOSE: Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification. PROCEDURES: For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol-1) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol-1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol-1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis. RESULTS: The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo 18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo 18F-FDG PET/MRI results. CONCLUSIONS: When taken together, our results demonstrate the feasibility of 18F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.