Pivotal roles of tumor-draining lymph nodes in the abscopal effects from combined immunotherapy and radiotherapy

Abstract

Background: Currently, due to synergy enhancement of anti-tumor effects and potent stimulation of abscopal effects, combination therapy with irradiation and programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint inhibition (immuno-radiotherapy, iRT) has revolutionized the therapeutic guidelines. It has been demonstrated that tumor-draining lymph nodes (TDLN) are essential for effective antitumor immunity induced by radiotherapy, immunotherapy, or iRT. Given that the function of TDLN in iRT remains unclear, this study aimed to investigate the function and mechanism of TDLN in iRT-induced abscopal effects.

Methods: The function of TDLN was evaluated using unilateral or bilateral MC38 and B16F10 subcutaneous tumor models with or without indicated TDLN. The flow cytometry, multiple immunofluorescence analysis, and NanoString analysis were utilized to detect the composition and function of the immune cells in the primary and abscopal tumor microenvironment. Additionally, we tempted to interrogate the possible mechanisms via RNA-sequencing of tumor-infiltrating lymphocytes and TDLN.

Results: TDLN deficiency impaired the control of tumor growth by monotherapy. Bilateral TDLN removal rather than unilateral TDLN removal substantially curtailed iRT-stimulated anti-tumor and abscopal effects. Furthermore, in the absence of TDLN, the infiltration of CD45⁺ and CD8⁺ T cells was substantially reduced in both primary and abscopal tumors, and the anti-tumor function of CD8⁺ T cells was attenuated as well. Additionally, the polarization of tumor-associated macrophages in primary and abscopal tumors were found to be dependent on intact bilateral TDLN. RNA-sequencing data indicated that impaired infiltration and anti-tumor effects of immune cells partially attributed to the altered secretion of components from the tumor microenvironment.

Conclusions: TDLN play a critical role in iRT by promoting the infiltration of CD8⁺ T cells and maintaining the M1/M2 macrophage ratio.

Keywords: T cells; immuno-radiotherapy; tumor-associated macrophage; tumor-draining lymph node.

FIGURE 1

TDLN are essential for anti‐tumor and abscopal effects induced by iRT. (A) Schematic diagrams of unilateral and bilateral subcutaneous tumor models. (B‐C) Tumor volumes of the unilateral MC38 (B) and B16F10 (C) tumor models treated as Panel A. (D) Schematic diagrams of FTY720 administration. (E‐F) Tumor volumes (E) and representative images (F) of isolated MC38 tumors treated with iRT with or without FTY720 treatment. (G) The proportion of CD3⁺ T cells in peripheral blood in mice treated with or without FTY720. (H) Tumor volumes of the bilateral MC38 and B16F10 tumor models treated as Panel A. Representative data were obtained from 2‐3 independent experiments. Data are expressed as mean ± SEM (n = 6‐9 per group).^*, P < 0.05; ^**, P < 0.01; ^****, P < 0.0001. Abbreviations: TDLN, Tumor‐ draining lymph nodes; PD‐1, programmed cell death protein 1; SEM, standard error of the mean; Con, control; Sham, sham surgery; Res, resection; RT, radiotherapy; iRT, combination of radiotherapy and anti‐PD‐1; Res‐Left, removal of TDLN on the left side; Res‐Right, removal of TDLN on the right side; Res‐Bilateral, removal of TDLN on both sides.

FIGURE 2

Immune response related genes and pathways enriched in tumors treated with iRT are altered in the absence of TDLN. (A) Heat‐map demonstrating differently expressed genes in TILs isolated from primary and abscopal tumors treated with iRT in the presence or absence of bilateral TDLN (based on RNA‐seq data). (B) Volcano plot demonstrated significantly differentially expressed genes in TILs isolated from primary or abscopal tumors treated with iRT in the presence or absence of bilateral TDLN. (C) KEGG pathway analysis (based on RNA‐seq data) showing the top 15 pathways upregulated in TILs isolated from primary and abscopal tumors subjected to iRT in the presence or absence of bilateral TDLN. (D) Venn diagrams demonstrated the top 15 upregulated pathways in the iRT vs. Con group and iRT vs. iRT + Res group of primary and abscopal tumors (n = 3 for each group). Abbreviations: TDLN, Tumor‐ draining lymph nodes; TIL, Tumor‐ infiltrating lymphocytes; iRT, combined immunotherapy and radiotherapy; Res, bilateral TDLN resection; iRT + Res, iRT with bilateral TDLN resection; RNA‐seq, RNA‐sequencing; FC, fold change; non‐sig, non‐significant.

FIGURE 3

TDLN removal abrogates iRT‐induced anti‐tumor immune responses. Primary and abscopal tumors were collected and subjected to flow cytometric analysis. Representative data and quantitative analysis of CD45⁺ cells (A) and CD8⁺ T cells (B) isolated from primary and abscopal tumors. Representative data and quantitative analysis of Granzyme B⁺ (C, E) and IFN‐γ⁺ (D, F) CD8⁺ T cells isolated from primary and abscopal tumors. Representative data are obtained from 2‐3 independent experiments. Data are expressed as mean ± SEM (n = 5‐6 per group). ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001. Abbreviations: TDLN, Tumor‐ draining lymph nodes; SEM, standard error of the mean; IFN‐γ, interferon‐γ; Con, control; Res, resection; iRT, combined immunotherapy and radiotherapy; iRT + Res, iRT with bilateral TDLN resection.

FIGURE 4

Indispensable role of CD8⁺ T cells in iRT‐induced abscopal effects relies on intact TDLN. (A) Multiplex immunofluorescence staining for quantitative analysis of CD45⁺ and CD8⁺ T cells isolated from primary and abscopal tumors of MC38 mouse models. (B, C) Tumor volumes (B), survival curve and tumor growth curve (C) of the bilateral MC38 models. Representative data are shown from two independent experiments. Data are expressed as mean ± SEM (n = 3‐8 per group). ^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001, ^****, P < 0.0001. Abbreviations: TDLN, Tumor‐ draining lymph node; SEM, standard error of the mean; Con, control; Res, resection; iRT, combined immunotherapy and radiotherapy; iRT + Res, iRT with bilateral TDLN resection.

FIGURE 5

TDLN maintains iRT‐mediated TAM polarization in the primary and abscopal tumor microenvironment. (A) Quantitative analysis of M1 and M2‐like macrophages isolated from primary and abscopal tumors treated with iRT in the presence or absence of bilateral TDLN. (B) Heat maps demonstrating genes related to M1 or M2 macrophages in primary and abscopal tumors (NanoString molecular analysis based on RNA‐seq data). (C) Representative images and quantitative analysis of M1‐like (F4/80⁺ iNOS⁺) and M2‐like (F4/80⁺ CD206⁺) macrophages in primary and abscopal tumors. Data are expressed as mean ± SEM (n = 3‐7 per group). ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. Abbreviations: TDLN, Tumor‐draining lymph node; TAM, tumor‐associated macrophage; SEM, standard error of the mean; Con, control; Res, resection; iRT, combined immunotherapy and radiotherapy; iRT + Res, iRT with bilateral TDLN resection.

FIGURE 6

Schematic diagram for explaining the role of TDLN in reprogramming the iRT‐induced tumor immune microenvironment in both primary and abscopal tumors. iRT lead to significant infiltration of functional CD8⁺ T cells and M1 TAM in both primary and abscopal tumors in the presence of TDLN, leading to efficient anti‐tumor effects. However, the absence of bilateral TDLN impaired the enrichment and cytotoxic capacity of CD8⁺ T cells coupled with M2 polarization of TAM in the primary and abscopal tumor microenvironment, resulting in compromised iRT‐induced tumor regression. Abbreviations: TDLN, tumor‐draining lymph nodes; TAM, tumor‐associated macrophage; iRT, combined immunotherapy and radiotherapy; iRT + Res, iRT with bilateral TDLN resection.