Abstract
Purpose This study is aimed at immunologically characterizing sentinel lymph nodes (SNs) in colorectal cancer (CRC) patients and identifying changes in immunological phenotype and function of SNs isolated from the tumor immunosuppressive microenvironment. Methods A total of 53 pairs of matched SNs and non-SNs (NSNs) were collected by using a lymph node tracer dye. Flow cytometry was performed to detect the immunophenotype of T cells as well as the expression of activation and inhibitory markers. Differential expression and distribution of characteristic immune cell markers were analyzed by multiplex immunohistochemistry (mIHC). Transcriptomics analysis was conducted to compare the differences in the expression of immune-related genes among lymph nodes. The ex vivo culture of lymph nodes was carried out to examine changes in immunological phenotypes and functions. Results Compared with NSNs, SNs harbored a significantly higher percentage of regulatory T cells (Tregs) but a lower proportion of MoMDSCs. As indicated in the mIHC assays, Tregs, T follicular helper (Tfh) cells, and M2 macrophages were mainly distributed in cortical areas, germinal centers, and subcapsular sinus areas, respectively, while significantly higher numbers of Tregs and Tfh cells were detected in SNs as compared to NSNs. Moreover, GSEA revealed that T cell activation genes and CD8+ T cell exhaustion-related genes are enriched in SNs and NSNs, respectively. The ex vivo culture led to an increase in the proportion of CD4+ cells, while activating T cells in SNs. In addition, SNs displayed a higher increase in the expression of cytokines IFN-γ, TNF-α, and sFas than NSNs. Conclusion SNs are shown to be in an immune active state in vivo, while highly expressing inhibitory cytokines and suppressive markers. The ex vivo culture enhanced antitumor immunological function of SN-T cells, providing a starting material for adoptive cell therapy for CRC.
Highlights
Colorectal cancer (CRC) is the third most common diagnosed cancer and the fourth most deadly malignancy, with almost 900,000 deaths annually worldwide [1, 2]
We previously showed that adjuvant sentinel lymph nodes (SNs)-T lymphocyte immunotherapy is safe and feasible for postoperative CRC patients, while improving longterm survival of metastatic CRC [13]
The subtypes of immunocytes present in the SN and corresponding nonsentinel nodes (NSN) were determined by flow cytometry
Summary
Colorectal cancer (CRC) is the third most common diagnosed cancer and the fourth most deadly malignancy, with almost 900,000 deaths annually worldwide [1, 2]. As a heterogeneous disease of the intestinal epithelium, CRC involves multiple factors and stages, which are characterized by the accumulation of mutations and immune response disorders [3]. While the clinical treatment of CRC has been dominated by surgical resection, chemoradiotherapy, and targeted therapy, these methods often fail to eradicate tumor foci, resulting in tumor recurrence, progression, and poor prognosis. Alternative strategies need to be developed for the treatment of CRC patients. The body’s immune system plays a role in immune surveillance, while it promotes the immune escape of tumors in certain stages, affecting tumor occurrence and development. After initial success in the treatment of melanoma, immunotherapy has been demonstrated as the most promising treatment option for various types of solid tumors, including CRC
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