Fig 1: PLX3397 and αCSF1R reduce TAM-derived CCL22 that affect T cell recruitment. (A) 15 days after treatment began, serum of mice in each group was collected and the CBA method was used to detect Th1/Th2/Th17-related inflammatory factors, n = 10. (B) 15 days after treatment began, serum chemokine levels of CCL2, CCL22, CXCL10 and CXCL9 were detected by ELISA kit, n = 10. (C) Serum levels of CCL22 at day 0,5,10,15 and 20 during the development of murine subcutaneous tumors, with treatment starting at day 7 after tumor inoculation, n = 10. (D) Macrophages, T cells and CD45− cells within the tumor on 9DPT and 15 DPT were sorted and the overall transcription levels of these cells were analyzed. (E) Representative flow cytometry histogram of CCR4 levels in Treg in tumor of LLC subcutaneous tumor model. (F) CD8T/Treg ratio at 13 DPT treated with CCL22 combined with PLX3397; And (G) tumor volume changes in during the treatment, n = 10.
Fig 2: The combination of αPD-1 and PLX3397 showed better therapeutic effect, and αPD-1 further promoted the polarization switch of TAM. (A) The change of tumor volume within 15 days after treatment began, n = 5. LLC subcutaneous tumor model was developed and the treatment experiment was conducted according to the description in the method; (B) Survival rate of different treatment groups after PLX3397 combined with αPD-1, n = 5; (C) The mice were sacrificed on 40 days after treatment began, the subcutaneous tumors were collected, photographed and (D) the tumors were weighed; (E) The proportion of CD8+ T cells/Treg cells was analyzed by flow cytometry at day 40 after treatment began; (F) One of two independent αPD-1 treatment experiments, n = 5, (G) the tumors of 40 DPT were weighed and (H) The proportion of CD8+ T cells/Treg cells was analyzed by flow cytometry. (I) Representative figure of FISH was used to identify Ccl22 transcription in tumor and (J) integrated optical density statistical results. (K) qPCR was used to analyze the transcription of Ccl22 in purified tumor macrophages on day 40 after treatment. (L) The transcriptional levels of Arg1 and Tgfb1 in separated TAM on day 15 after treatment began were analyzed by qPCR.
Fig 3: ZEB1 in macrophages has no major direct effect on tumor cells but is important for CCL2- and CCL22-driven recruitment of CD8 + T cells.a Confluence of KPC cells alone or co-cultured with LysMCtrl or LysMΔZeb1 BMDMs (n = 3). b Representative images at t = 28 h and quantification over time of KPC cell invasion into a scratch wound without or with co-culture of LysMCtrl or LysMΔZeb1 BMDMs (n = 3). c Confluence of KPC cells alone or with LysMCtrl or LysMΔZeb1 BMDM conditioned medium (CM) (n = 2 KPC CM, n = 3 LysMCtrl and LysMΔZeb1 CM). d Transwell migration assay of CD8 + T cells alone or towards LysMCtrl or LysMΔZeb1 BMDMs in absence or presence of recombinant CCL2 and CCL22 (left panel, n > 3) or absence or presence of anti-CCL2 and anti-CCL22 antibodies (right panel, n = 3). Means ± SD; *p < 0.05; **p < 0.01; ns: not significant; 2-way ANOVA.
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