Background
As a local minimally invasive treatment, microwave ablation (MWA) has been widely used in the treatment of many solid tumors [
1,
2]. MWA can kill cells, which uses electromagnetic waves to generate heat and trigger antigen release and even immune responses [
3]. MWA has been shown to offer various advantages over other types of thermal ablation across studies [
4]. MWA causes higher volumes of necrosis, higher ablation rates, and increased homogeneity of the decaying region, allowing it to vaporize larger lesions [
5,
6]. Several studies have also shown that MWA combined with immune therapy can stimulate a robust anti-tumor immune response and has promising results [
3,
7‐
11]. For example, it has been suggested that in the tumor model established by 4T1 breast cancer cells, the MWA can activate the T-cell immune response, and the combination therapy based on MWA can significantly induce the Th1-type anti-tumor response [
10]. Moreover, we have previously confirmed that MWA combined with TIGIT blockade exerts synergistic effects against cancer in contrast to MWA or TIGIT blockade alone [
12].
Immune checkpoint inhibitor (ICI)-based immunotherapies have demonstrated broad advantages and durable clinical outcomes in cancer treatments [
13‐
15]. However, only a subset of patients acquires substantive benefits from immunotherapy. This discrepancy suggests that it is urgently necessary to reveal the underlying mechanism. LAG3, also called CD233, is an inhibitory receptor that is highly expressed on activated T cells, natural killer (NK) cells, and plasmacytoid dendritic cells (DCs)[
16‐
18]. LAG3-targeted immunotherapies have been tested as an important anti-tumor agent in lots of clinical trials for multiple types of cancer [
19]. It goes either as a single blockade strategy or in combination with other marketed ICIs. For example, the recent clinical trial (NCT03470922) has reported that a combination of LAG-3 and PD-1 blockade can provide a greater benefit with regard to progress-free survival (PFS) than PD-1 blockade alone in patients with previously untreated metastatic or unresectable melanoma [
15]. Despite its late stage in clinical trials, the role of LAG3 in the immune cellular network has not yet been fully addressed. Our previous studies have shown that radiofrequency ablation (RFA) combined with PD-1 blockade, or MWA combined with TIGIT blockade, can serve as important combinational therapeutic strategies [
9,
12]. Moreover, we have also analyzed publicly available single-cell RNA-sequencing (scRNA-seq) data from the pancreatic ductal adenocarcinoma (PDAC) mouse model with and without RFA therapy, and found that LAG3 expression is up-regulated in CD8
+ T and CD4
+ T cell subsets after RFA, suggesting the potential possibilities for the design of combination therapeutic strategy [
20].
In the present study, we treated mice with MWA or LAG3 blockade and phenotyped their anti-tumor immune responses in a mouse colon cancer MC38 model. We found that MWA could greatly induce the expression of LAG3 on tumor-infiltrating lymphocytes (TILs) in MC38 tumors. These findings suggested that LAG3 expression, which was up-regulated as an immune self-restrictive molecule after T cell activation, could play a crucial role in combination therapy with MWA. Meanwhile, in the MC38 tumor model, introducing LAG3 blockade to MWA extended survival and postponed tumor development. By attracting CD8+ TILs penetrating the tumor microenvironment (TME), LAG3 blockade combined with MWA increased the proliferation and activities of CD8+ T cells while reshaping myeloid cells. These findings supported the idea that LAG3 blockade combined with MWA might be a unique treatment regimen that improved anti-tumor immunity synergistically.
Material and methods
Cell line and mice
The MC38 cells (mouse colon cancer cell line) in the present study have been used in our previous report [
12]. MC38 cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cavens Laboratory Animals provided the 6–8 week-old C57BL/6 mice (male) and kept them in a particular pathogen-free (SPF) facility (Changzhou, China). All animal studies were carried out in accordance with protocols authorized by the Third Affiliated Hospital of Soochow University’s Ethics Committee.
Animal models, MWA treatment and LAG3 blockade therapy
Each C57BL/6 mouse had a total of 3 × 10
6 MC38 tumor cells implanted subcutaneously into their bilateral sides (1.5 × 10
6 MC38 cells for each side) to establish the tumor model according to our previous report [
12]. Only after the tumor volume reached roughly 300 mm
3 was MWA can be performed on the tumor on the right flank. MWA was conducted using an ablation electrode (Microwave Ablation Antennas, Canyon Medical Inc., Jiangsu Nanjing) percutaneously inserted in the center of the tumor as reported in our previous study [
12]. The treatments lasted 2–4 min at 70 °C and 8 W. LAG3 blockade was then intraperitoneally administered four times every 3 days, starting on day 1 after MWA. An anti-LAG3 mAb (Clone C9B7W, BioXcell, USA) was administered at a dose of 200 μg per mouse per injection. The diameters of the tumors on the left flank were measured every other day, and the tumor volume was calculated using the formula as follows: Volume = Length × Width
2/2. Tumor growth curve and the overall survival (OS) were observed and charted.
Flow cytometry
Tumor tissues from the left tumors were collected and digested with Liberase TL (Catalog No. 05401020001, Roche) and DNase I (Catalog No. 10104159001, Roche) for 30 min at 37 °C, and prepared into single-cell suspension as reported in our previous study [
12]. Antibodies used in staining and flow analysis included CD45 (Clone 30-F11), CD3 (Clone 17A2), CD4 (Clone GK1.5), CD8 (Clone 53–6.7), NK1.1 (Clone PK136), LAG3 (Clone C9B7W), Foxp3 (Clone 3G3), CD11b-BV650 (Clone M1/70), MHC-II (Clone AF6-120.1), CD11c (Clone N418), CD206 (Clone C068C2), F4/80 (Clone BM8) and CD103 (Clone M290). Intracellular cytokine staining was also performed as described in previous study [
12]. Following stimulation, the cells were labeled with antibodies against surface markers, fixed, and permeabilized using the Invitrogen Fixing/Permeabilization Solution kit’s manufacturer’s instructions. Antibodies against TNF-α (Clone MP6-XT22) and IFN-γ (Clone XMG1.2) were used to stain the fixed cells. A BD FACS Aria II flow cytometer was used to collect data, which were then analyzed using FlowJo software.
scRNA-Seq
The procedure described above was used to make single-cell suspensions of tumor cells from the left tumors. The cells were enriched for FACS sorting using the CD45 (TIL) Microbead Mouse Kit (Catalog No. 130-110-618, Miltenyi Biotec, Leiden, the Netherlands) and labeled with the antibodies Ghost DyeTM Violet 510 Viability Dye (Cell Signaling Technology) and Percp-Cy5.5-CD45 (Clone 30-F11). A BD Aria II device was used to sort about 5 × 105 CD45+ cells per sample. Single cells were sorted into flow tubes based on the FACS analysis, and cell viability was determined by calculating the AOPI to guarantee adequate cell quality. The cell suspension was then put onto the chromium single-cell controller (10X Genomics) to form single-cell gel beads in the emulsion according to the manufacturer’s directions, with 300–600 viable cells per microliter as measured by CountStar. An S1000TM Touch Thermal Cycler (Bio-Rad) was used to perform single-cell transcriptome amplification at 53 °C for 45 min, followed by incubation at 85 °C for 5 min and storage at 4 °C. The quality of the cDNA templates was tested using Agilent 4200 equipment after they were produced and amplified (performed by CapitalBio Technology, Beijing).
scRNA-Seq data processing, integration of multiple scRNA-Seq data, dimension reduction, and unsupervised clustering
The Cell Ranger Single-Cell Software Suite was used to match the freshly generated scRNA-seq data acquired from 10X Genomics to the mm10 mouse reference genome and quantify it. The pre-filtered data obtained by Cell Ranger was used to construct a Seurat object with the R package Seurat (version 3.2.3). With the DoubletFinder package, doublets were eliminated. The overall UMI count, the number of identified genes, and the fraction of mitochondrial gene count per cell were all used to apply quality control to cells in a stepwise way. Cells with more than 5,000 UMI counts and 10% mitochondrial gene counts were specifically screened out. The workflow in Seurat was used to analyze single-cell data for dimension reduction and unsupervised clustering analysis. Using the Find Variable Features function with the option “n features = 2,000,” 2,000 highly variable genes were chosen for downstream analysis. The data were then integrated and a new matrix with 3,000 features was created, in which the possible batch effect was regressed, using the Integrated Data function. To minimize the dimensionality of the scRNA-seq dataset, principal component analysis (PCA) was conducted on an integrated data matrix. The top 40 PCs were submitted to downstream analysis using Seurat’s Elbow plot program. The primary cell clusters were found using Seurat’s Find Clusters tool with a resolution of 0.1. The clusters were then displayed using two-dimensional tSNE or UMAP plots. Each cell was classified into a recognized biological cell type using conventional markers established in a previous work.
Differential gene expression analysis
DEGs were identified between clusters using the EdgeR package (version 3.28.1). The calcNormFactors function was used to normalize the raw data from the Seurat object using TMM (trimmed mean of M-values), and the estimateDisp function was used to estimate the dispersion of gene expression levels. The DEGs were chosen for display using the Seurat package’s DotPlot function.
Trajectory analysis
Single-cell trajectories were built with the Monocle2 R package (version 2.14.0) that introduced pseudotime. Genes were filtered by the following criteria: expressed in more than 10 cells. Then, the ECDF plot was performed by the ggplot2 package geom_ecdf() function to compare different states between two samples.
Statistical analysis
Statistical analysis was performed using Graphpad Prism v8. The log-rank test was used for comparisons in overall survival. Two-way ANOVA was used for comparing tumor growth curves. The two-tailed un-paired Student’s t-test was used to compare two groups and the ANOVA test was used for multiple comparisons.
Discussion
A great deal of success has been achieved in the treatment of various malignancies with combination immunotherapy based on ICI [
23,
24]. In many preclinical and clinical settings, ablation coupled with ICIs has produced unprecedented OS benefits for cancer patients. Shi et al. demonstrated that RFA could induce robust T cell-mediated anti-tumor immune responses in distant tumors, and Chen et al. have also shown that combination therapy of MWA and TIGIT blockade synergistically promotes anti-tumor immune response in mouse colon cancer model [
9,
12]. Furthermore, RFA does not prevent tumor recurrence in certain individuals, indicating that alternative immune-suppression pathways were involved in the TME after FRA [
25]. Many clinical trials comparing RFA and MWA have demonstrated that both two methods represented similar efficacy and safety, while MWA has technical advantages in terms of reduced heat sink effect and faster ablation [
4,
26]. In the present study, we found that MWA can greatly induce the expression of LAG3 on TILs, especially on CD8
+ TILs, indicating that the combination regimen could provide benefits. Therefore, it’s of great imporatance to reveal the therapeutic effect of combinational strategy against cancer and explore its possible clinical application prospect.
As we know, LAG3 is highly expressed on activated T cells, NK cells, and plasmacytoid DCs [
16‐
18]. Similar to the CD4 molecule, LAG3 has four distinct Ig-like domains, sharing the same ligand as CD4, the MHC-II [
27]. LAG3 blockade results in enhanced T cell expansion and up-regulated function signature in vitro [
28,
29]. Upon tumorigenesis and chronic viral infection, such as the LCMV C13 strain, LAG3 is up-regulated in T cells along with other immune inhibitory receptors, such as PD-1, TIM3, and TIGIT [
30‐
32]. Moreover, currently, as the next generation of immune checkpoint therapy in cancer, LAG-3 has been confirmed as an important candidate target, for example, the clinical trials NCT00732082, NCT00349934, NCT02614833, NCT01968109 and NCT02460224, have shown that LAG-3 blockade can not only improve the antitumor immune responses but also can potentiate other forms of immunotherapy [
33]. However, the molecular mechanism of how LAG3 affects the T cell function in the scenario of cancer still remains to be illustrated. It has been suggested that the antagonistic mAb against LAG3 could blockade the interaction between LAG3 and MCH-II molecules expressed by tumor and/or immune cells, leading to the promotion of tumor cell apoptosis [
34]. Another phase I clinical trial has reported that, IMP321, a soluble form of LAG3, can have an objective response rate (ORR) of 50% in metastatic breast cancer when combined with paclitaxel [
35]. Therefore, we aimed to identify whether LAG3 blockade could significantly enhance the MWA-induced anti-tumor immune response by introducing more inflamed tumors and more functional CD8
+ T cells.
In summary, MWA dramatically induced the expression of LAG3 on different TILs sub-populatios in TME, and anti-LAG3 treatment in combination with MWA, could significantly suppress tumor development, increase effector CD8
+ TILs, and restore the tumor-killing function of exhausted CD8
+ T cells. The present similar mechanism was also found in our previous studies, such as RFA combined with PD-1 blockade, and MWA combined with TIGIT blockade, it is necessary to evaluated the therapeutic efficacy of the above three combined treatment methods separately, or even perform the investigation of triple therapy strategy, such as MWA plus PD-1 and LAG3 blockade [
9,
12]. In fact, we tried MWA plus TIGIT and PD-1 blockade, and the results revealed that the triple therapy showed significant advantages in contrast to MWA plus TIGIT, MWA plus PD-1, or even MWA and TIGIT alone (data not shown). Furthermore, the LAG3 blockade plus MWA dramatically improved T cell interaction, demonstrating that the combination of LAG3 blockade and MWA could effectively suppress the inhibitory signals on T cells in a synergistic manner. Therefore, LAG3 blockade combined with MWA might be employed in the clinical setting to reprogram the TME in an anti-cancer manner, revealing the potential value of the clinical application.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.