Panoply™ Human CCR8 Knockdown Stable Cell Line

Factors released by glioma-associated microglia/macrophages (GAMs) play a crucial role in the progression of glioblastoma multiforme (GBM). Here, researchers investigated the importance of CCL18, a cytokine expressed in human but not rodent GAMs, as a regulator of glioma growth. Because CCL18 signaling cannot be studied in classic mouse glioma models, the researchers developed a method to transplant induced pluripotent stem cell-derived human microglia and human glioma cells into mouse brain slices depleted of resident microglia. They observed that CCL18 promoted glioma cell growth and invasion. Chemokine (C-C motif) receptor 8 (CCR8) has been identified as a functional receptor for CCL18 on glioma cells, while ACP5 (acid phosphatase 5) has been revealed as a key component of the downstream signaling cascade mediating glioma growth. Based on results from in vitro, ex vivo humanized glioma models, and in vivo GBM models, the researchers concluded that microglia/macrophage-derived CCL18 promotes glioma growth.

To test whether CCR8 serves as a functional receptor for CCL18 in glioma cells, the researchers stimulated glioma cell proliferation with CCL18 and added a CCR8-neutralizing antibody. The CCL18-induced increase in proliferative activity, as measured by the CCK-8 assay, was significantly attenuated by the CCR8-neutralizing antibody (Figure 1C). For further functional testing, they measured tumor growth in mouse microglia-depleted obstructive sclerosis (OBS) inoculated with U251MG, LN229, and patient-derived glioma cells. The CCR8-neutralizing antibody abolished the CCL18-induced increase in tumor growth (Figure 1D). Next, the researchers inoculated CCR8-knockdown glioma cells (U251MG and LN229) into OBS. CCL18 treatment did not increase tumor growth, a phenomenon observed in control glioma cells (Figure 1E). Furthermore, iMGL was co-inoculated with glioma cells into OBS and tumor growth was monitored. While co-injection of iMGL increased tumor growth in control glioma cells, this effect was abolished in CCR8-knockdown glioma cells (Figure 1F). To further validate this effect in vivo, researchers knocked down CCR8 in U251MG cells, inoculated these cells into the brains of nude mice, and treated tumors with CCL18. In CCR8-deficient tumors, CCL18 treatment had no effect on survival (Figure 1G), while knockdown of CCR8 alone did not alter glioma growth (Figure 1G). Furthermore, blocking CCR8 by adding a CCR8-neutralizing antibody successfully attenuated the effects of CCL18 (Figure 1H). Moreover, tumor volumes were also assessed in vivo by subcutaneously injecting CCR8 knockdown glioma cells (U251MG) with THP-1 macrophages. Tumors arising from CCR8-knockdown glioma cells were significantly smaller (Figure 1I).

Figure 1. CCR8 on tumor cells acts as functional receptor for CCL18. (Huang Y, et al., 2022).