A Novel Peptide Suppressing M2 Polarized Tumor Associated Macrophages Enhances Tumor Response to Chemotherapy
Authors: GHEBREMEDHIN ANGHESOM A , Jones‐Trich Jacqueline , Karanam Balasubramanyam , Addu‐Addai Benjamin , Garvin Charles , Martin George , Lopez Henry , Jaynes Jesse , Yates Clayton 
Published: November 2023

IntroductionMultiple reports have demonstrated that M2‐polarized tumor associated macrophages (TAMs) play a significant role in promoting tumor metastasis, survival and resistance to chemotherapy. Thus, limiting the pro‐tumorigenic activity of TAMs may hold great therapeutic promise.ObjectiveTo determine whether targeting Cluster of Differentiation 206 (CD206) receptor on M2 cells or TAMs with a peptide having both CD206 specificity and apoptotic activity, will limit the protumorigenic effects of TAMs and lead to more effective clearance of cancer cells than chemotherapy alone.MethodAn artificial peptide (10mers) with a striapathic arrangement of hydrophobic and hydrophilic amino acids was designed to specifically target CD206. Primary cells were isolated from C57BL/6J mice from Jackson Laboratory and were polarized to M1 and M2 phenotypes by treating with IL‐4 (20ng/ml) and IFN‐γ (10ng/ml) respectively from R&D Biosciences for 24 hrs. CD206 targeting peptide was biotin‐labeled and utilized to treat M2‐polarized cells. Precipitated proteins were analyzed by mass spectrometry analysis, and informatics software. In vitro proliferation and confirmation of apoptosis was assessed using Annexin/PI staining and Western blot analysis for Caspase 8 expression and activity. In xenograft models of triple negative breast cancer (MDA‐MB‐231), mice were treated with CD206 peptide and Gemcitabine, singly or in combination. Immunohistochemical analysis of treated tumors was also performed to assess the expression of macrophages.ResultsMass spectrometry analysis of gel bands from the biotin/streptavidin pull down assay confirmed CD206 as a cell surface target. Bioinformatics analysis of the proteomics data from the pull down assays further confirmed that apoptotic proteins were significantly more highly expressed in the M2 populations than in the M1 populations. Treatment with the peptide demonstrated a significant decrease in cell proliferation only in M2 macrophages. FACS analysis of Annexin/PI staining and Caspase 8 activity, demonstrated by Western blot, showed that CD206 targeting peptide caused apoptosis specifically in M2 macrophages. In xenograft models of triple negative breast cancer (MDA‐MB‐231) mice treated with peptide alone (10 mg/kg qD subcu), there was a 39% reduction in MD‐MB‐231 tumor growth; Gemcitabine @ 40 mg/kg q4D demonstrated a 68% reduction; however, peptide plus Gemcitabine completely blocked tumor growth. Moreover, immunohistochemistry staining of treated tumors showed a decrease in the expression of CD206 positive M2 macrophages and CD45/CD25 positive Treg cells, and an increase in CD86 positive M1 macrophages. These results were consistent with the in vitro results obtained.Discussion and ConclusionThese results suggest that a targeting CD206 peptide engineered to promote apoptosis, enhances the tumor response to chemotherapy by limiting the pro‐tumorigenic activity of TAMs.Support or Funding Information‐ This work is supported by the MSM/TU/UAB Comprehensive Cancer Center Partnership [NCI]: Tuskegee University – U54 CA118623‐ Riptide Biosciences, Inc