TRIM37 Promotes Pancreatic Cancer Progression through Modulation of Cell Growth, Migration, Invasion, and Tumor Immune Microenvironment

Abstract

TRIM37 dysregulation has been observed in several cancer types, implicating its possible role in tumorigenesis. However, the role of TRIM37 in pancreatic cancer progression remains unclear. In the present study, we observed that TRIM37 knockdown resulted in reduced proliferation, clonogenicity, migration, and invasion ability of pancreatic cancer cells. Furthermore, an in vivo study using an orthotopic syngeneic animal model further confirmed that reduced expression of TRIM37 in cancer cells suppressed tumor growth in vivo. Moreover, in mice bearing TRIM37 knockdown pancreatic cancer cells, the proportion of CD11b⁺F4/80⁺MHCII^low immunosuppressive macrophages was significantly reduced in tumor milieu, which might be due to the regulatory role of TRIM37 in cytokine production by pancreatic cancer cells. Collectively, these findings suggest a key role of TRIM37 in promoting pancreatic cancer progression.

Keywords: TRIM37; immune microenvironment; pancreatic cancer.

Figure 1

Knockdown of TRIM37 decreased pancreatic cancer cell viability. An MTT assay was performed to measure the viability of cells at different time points. The numbers represent the mean absorbance at 570 nm ± SEM (n = 3). Statistical significance was determined using two-way ANOVA with Bonferroni’s post-hoc test (p < 0.001: *** compared to shLuc). WT: wild type cells; shLuc: cells bearing shLuc plasmid; shTRIM37-1: cells bearing shTRIM37-1 plasmid; shTRIM37-2: cells bearing shTRIM37-2 plasmid.

Figure 2

The knockdown of TRIM37 reduced pancreatic cancer cell migration ability. A wound healing assay was conducted to measure pancreatic cancer cell migration ability of Pan18, BxPC-3, and PANC-1 cells. Representative images of different pancreatic cancer cell monolayers (Original magnification, ×10) and quantification of wound closure are presented. Numbers represent the mean wound closure (migration) ± SEM (n = 3). Statistical significance was determined using two-way ANOVA with Bonferroni’s post-hoc test (p > 0.05: ns, p < 0.01: **, p < 0.001: *** compared to shLuc).

Figure 2

The knockdown of TRIM37 reduced pancreatic cancer cell migration ability. A wound healing assay was conducted to measure pancreatic cancer cell migration ability of Pan18, BxPC-3, and PANC-1 cells. Representative images of different pancreatic cancer cell monolayers (Original magnification, ×10) and quantification of wound closure are presented. Numbers represent the mean wound closure (migration) ± SEM (n = 3). Statistical significance was determined using two-way ANOVA with Bonferroni’s post-hoc test (p > 0.05: ns, p < 0.01: **, p < 0.001: *** compared to shLuc).

Figure 3

The knockdown of TRIM37 reduced pancreatic cancer cell invasion ability. The invasions were measured using Matrigel invasion assay. Pan18, BxPC-3, and PANC-1 cells (5 × 10⁴) were seeded in the upper chamber for 2 days, and then the invaded cells were counted. Representative images of invaded pancreatic cancer cells (Original magnification, ×20) and quantification of invaded cells are presented. The numbers represent the mean invading cell count ± SEM (n = 3). Statistical significance was determined using two-way ANOVA with Bonferroni’s post-hoc test (p < 0.001: *** compared to shLuc).

Figure 4

The knockdown of TRIM37 inhibited the clonogenicity of pancreatic cancer cells. Colony formation assay was conducted by seeding 300 cells of Pan18 for 7 days, 500 cells of BxPC-3 for 7 days, and 1000 cells of PANC-1 for 18 days. Representative images of pancreatic cancer colonies and their quantification are presented. The numbers represent the mean colony count ± SEM (n = 3). Statistical significance was determined using one-way ANOVA with Bonferroni’s post-hoc test (p < 0.001: *** compared to shLuc).

Figure 5

The knockdown TRIM37 of pancreatic cancer suppressed tumor growth in vivo. Pan18-GFP-Luc cells (5 × 10⁴) were injected orthotopically into syngeneic C57BL/6 mice. Tumor growth was monitored using the IVIS spectrum system. (A) Images of pancreatic cancer bioluminescence signal on day 15 and quantification of signals during tumor progression. The numbers represent the mean total flux (p/s) (WT: n = 6, shLacZ: n = 5, shTRIM37-1: n = 6, shTRIM37-2: n = 5). (B) After 17 days, the mice were sacrificed, and the tumors were dissected and weighed. The numbers represent the mean tumor weight ± SEM (WT: n = 6, shLacZ: n = 5, shTRIM37-1: n = 6, shTRIM37-2: n = 5). Statistical significance was determined using one-way ANOVA with Bonferroni’s post-hoc test (p < 0.01: **, p < 0.001: *** compared to WT or shLacZ).

Figure 6

TRIM37 modulated cytokine production by pancreatic cancer cells in vitro. Pan-18 cells (3.5 × 10⁴) were seeded on 6-well tissue culture plates. After the attachment of the cells to the plate, the medium was replaced with fresh medium, and the cells were cultured for another 48 h. The cytokine concentration in the culture medium was measured by using Bio-Plex^TM Pro Mouse cytokine 23-plex panel (BioRad, Hercules, CA, USA). The numbers represent the mean ± SEM (WT: n = 3, shTRIM37: n = 3). Statistical significance was determined using the unpaired Student’s t-test (p < 0.05: *, p < 0.01: **, ns: not significant compared to WT).

Figure 7

TRIM37 modulated the immune system by increasing the proportion of CD11b⁺F4/80⁺MHCIIlow macrophages in the tumor milieu. Single-cell suspension was isolated from the tumor mass obtained from different groups, and flow cytometry was conducted to analyze the cell population. (A) Representative images of the gating strategy of CD11b⁺F4/80⁺MHCIIlow and CD11b⁺F4/80⁺MHCIIhi populations. (B) Quantification of mean fluorescence intensity of MHC class II on CD11b⁺F4/80⁺ macrophages (left), percentage of CD11b⁺F4/80⁺MHCIIlow macrophage population (middle), and percentage of CD11b⁺F4/80⁺MHCIIhi macrophage population. The numbers represent the mean ± SEM (WT: n = 3, shLacZ: n = 3, shTRIM37: n = 5). Statistical significance was determined using the unpaired Student’s t-test (p < 0.05: *, p < 0.01: ** compared to WT or shLacZ).