JMBD

More than 80% of lung cancer patients have non-small cell lung cancer (NSCLC), yet the five-year relative survival rate is low [1]. As a result, lung cancer is one of the leading causes of cancerrelated deaths in both men and women worldwide. In recent years, with the emergence of new therapies such as targeted therapy and immunotherapy, it has brought a new dawn of treatment to some patients [2]. Neoadjuvant immune checkpoint inhibitors (ICIs) have improved the clinical benefit of some lung cancer patients, but there is still a lack of precise biomarkers to screen out those who can benefit [3].

DCs are the most powerful antigen-presenting cells (APCs) in the body, and as a key component of the immune system, DCs play a vital role in the initiation and regulation of the immune response [4]. They are the most powerful antigen-presenting cells in the immune system, capable of ingesting, processing, and presenting antigens, activating naïve T cells, and initiating adaptive immune responses [5]. With the deepening of research, more characteristics and functions of DC cells have been gradually revealed, bringing new opportunities for the treatment and prevention of diseases. Studies have shown that factors such as the number and functional status of DCs and their presentation efficiency to lung cancerrelated antigens are closely related to the efficacy of lung cancer treatment [6]. It has been reported in the literature that DCs have made some progress in the therapeutic efficacy of lung cancer [7]. At the same time, there are also new discoveries in exploring the mechanism of the influence of the lung cancer microenvironment on DCs function, which is helpful to solve the problem of inhibition of DCs function [8]. We tested the proportion of DCs in lung cancer tissues to verify the correlation between DCs and the efficacy of immunotherapy.

Methods

Flow cytometry antibodies and other reagents

Fluorescent antibody cluster of differentiation (CD)3, CD19, CD11c, purchased from BD in the United States: CD45, Human leukocyte antigen (HLA)-DR were purchased from Biolegend: LIVE/DEAD Fixable Blue Dead Cell Stain (L/D) and BV stain buffer were purchased from Sigma-Aldrich; Fetal bovine serum (FBS, 500 mL, 319801-5) was purchased from ZETA LIFE: Collagenase type IV (50 U/mL, 17104-019) was purchased from Thermo Fisher; DNase I, 20 U/mL, 10104159002 was purchased from Roche.

Sample collection

Thirty-two lung cancer samples were taken from West China Hospital of Sichuan University, Of these, 11 were in the TN group, 8 in the MPR group and 13 in the non-MPR, and the pathological diagnosis of lung cancer patients was NSCLC, see (Table 1) for clinical information. All subjects signed the informed consent form. Surgically excised human NSCLC tissue, the sample was removed from the necrotic tissue, the blood and other impurities were flushed out in PBS, placed in 10% FBS+RPMI1640, and shipped back at 4°C. The whole process is completed within 2 hours.

Table 1: Clinical information for patients.

Sample processing

After the tissues were retrieved, 2 mL of 10% FBS + RPMI1640 was prepared in a 10 cm Petri dish on ice, the tissues were placed in the medium, the tissue blocks were fan cut into 4 parts in the center, each part was about 3 mm×3 mm×3 mm, and the grouping started to be processed and sheared in the process in 2 h, and the corresponding digestive enzymes were added into each group respectively, and the digestion was rotated in an incubator at 37 ℃ for 30 min. After completion of digestion, centrifuge at 100 g for 1 min at 4 ℃, remove the undigested tissue to the bottom of the tube, and aspirate the supernatant. After digestion, add 1 times the volume of 2% FBS + RPMI1640 to terminate the digestion, centrifuge at 300 g for 7 min at 4 ℃, remove the supernatant. 3 mL of 2% FBS + RPMI1640 were washed, centrifuged at 300 g for 7 min at 4 ℃, and the supernatant was removed. 1mL of 0.04% BSA was resuspended the precipitate, filtered through a 70 μm screen, and the original tube was washed and the residual cells were sieved through a sieve with 1mL of 0.04% BSA. Sieve. The cell suspension of each group of cells was centrifuged at 300 g for 5 min at 4 ℃ and resuspended with an appropriate amount of 0.04% BSA. After the cell suspension was counted, the cell suspension was adjusted to 1.0×107 cells/mL and set aside.

Flow cytometry assay

A total of 32 human NSCLC tissue samples were detected and analyzed according to the established flow-through protocol. Twenty fluorescent antibodies and L/D dye were mixed in 50 μL PBS according to the titrated concentration, and 50 μL cell suspension was added and mixed thoroughly, incubated for 15 min at room temperature and protected from light, 1 mL PBS was added, centrifuged at 400 g for 5 min, washed, and the supernatant was discarded, resuspended in 300 μL PBS, and detected on the machine (Symphony A5, USA, BD Bioscience).

Data analysis

At least 106 living Events were collected and Flowjo 10.10.0 software was used for flow data analysis. Graphpad 8.0 statistical software was used for data processing and analysis. Measurements were expressed as x±s. Comparisons between groups were made using oneway ANOVA. Differences were considered statistically significant at P<0.05.

Results

Figure 1: Flow analysis and gating logic setting.

The MPR group had the lowest proportion of DCs

Through flow data analysis, it was found that the proportion of DC cells in MPR group was lower than TN group and non-MPR group, non-MPR group was higher than TN group, MPR group was the lowest and the highest in non-MPR group, as shown in (Figure 2).

Figure 2: The portion of DCs in three groups of samples in flow scatter plot. (A). Proportion of the DCs in the TN group. (B).MPR The proportion of the group DCs. (C). Proportion of the DCs in the non-MPR group.