Immature tertiary lymphoid structure formation was increased in the melanoma tumor microenvironment of IKZF1 transgenic mice

Background: IKZF1 promotes the occurrence of lymphoma and is also related to the development of breast cancer, liver cancer, and ovarian cancer. It was hypothesized that IKZF1 influences tertiary lymphoid structures (TLSs) formation and development in the tumor immune microenvironment, and this effect of IKZF1 on the tumor immune microenvironment has not been explored. Using melanoma grafts as a model, we investigated the effect of IKZF1 on the immune microenvironment of melanoma.

Methods: The Cell Count Kit-8 (CCK8) assay was used to detect the effect IKZF1 overexpression in melanoma cells on cell proliferation. The IKZF1 overexpression vector was constructed by homologous recombination. After linearization, the overexpression vector was microinjected into the fertilized egg. Transgenic mice overexpressing IKZF1 were screened by tail identification. After melanoma B16 mouse cells were digested into single cells, the tumor was subcutaneously implanted in C57BL6/J-wild type (WT) mice and IKZF1 transgenic mice, and the tumor growth of the 2 groups was compared. The number of TLSs in the tumor tissues of mice was analyzed after hematoxylin-eosin (HE) staining.

Results: Overexpression of IKZF1 in melanoma cells did not affect cell proliferation. The IKZF1 overexpression vector pcDNA3.1-CAG-IKAROS was successfully constructed. Viable fertilized eggs were obtained after microinjection. Transgenic mice stably expressing IKZF1 were identified by polymerase chain reaction (PCR). Compared with WT mice, the tumor load of IKZF1 transgenic mice increased significantly. HE staining showed that the number of immature TLSs in melanomas of IKZF1 transgenic mice increased significantly.

Conclusions: IKZF1 does not affect the proliferation of melanoma cells. Transgenic mice overexpressing IKZF1 were successfully constructed. IKZF1 is a key driver gene of the formation of immature TLS.