Folate Receptor Beta as a Marker of Immunosupressive Myeloid Derived Suppressor Cells and Tumor Associated Macrophages in the Tumor Microenvironment

Immunosuppression within a tumor environment is a key component of the development and ultimately spread of cancer in the body. Myeloid Derived Suppressor Cells (MDSC) and Tumor Associated Macrophages (TAMs) are two of the primary forces behind immunosuppression within the tumor environment. MDSC are a functionally defined group of myeloid cells that have the capacity to suppress anti-tumor T cell responses, induce regulatory T cells (Tregs), polarize inflammatory macrophages to TAMs, and promote metastasis. This broad ability to control the immune response has led to a great deal of focus on the function of these cells. However, despite efforts to characterize and target these cells, it remains difficult to identify them with MDSC isolated from the tumor and peripheral sites studied interchangeably. MDSC are divided into two subsets, monocyte like MDSC (M-MDSC) and granulocyte like MDSC (G-MDSC). To overcome the limitations in identification, we utilized folate receptor beta (FRβ) as a marker to isolate MDSC. Only MDSC isolated from the tumor site possessed a functional FRβ that could take up folate conjugates. Spleen isolated MDSC did not express FRβ. Currently, FRβ has been used as a marker of TAMs for delivery of folate conjugated drugs. In this study, it was determined that FRβ marks a population of highly functional TAMs and M-MDSC that are present only at the tumor site. Interestingly, only under hypoxic conditions does FRβ segregate with suppressive function. Additionally, FRβ segregates with iNOS expression and nitric oxide (NO) production as the key suppressive mechanism under hypoxic conditions in both M-MDSC and TAMs. This is an interesting observation considering that PD-L1 has been characterized as the primary suppressive mechanism of MDSC under hypoxic tension. Inhibition of PD-L1 by blocking antibody did not impair M-MDSC suppressive function and only moderately reduced TAM suppression in the FRβ+ populations. When utilizing an anti-FRβ antibody, FRβ could be targeted in an inflammatory system to deplete MDSC only at the tissue site and not in peripheral sites. However, in the MB49 solid tumor model the anti-FRβ antibody was unable to target MDSC or TAMs. This suggests limitations to antibody depletion through FRβ but does not rule out future applications of small molecule FRβ targeted drugs. It is not immediately clear how FRβ impacts suppressive function or if it is simply a maker that segregates with suppressive populations, which led us to utilize RNAseq to analyze FRβ positive and negative populations within M-MDSC and TAMs to identify pathways that segregate with FRβ. The IL-6 pathway was identified to segregate with FRβ in both M-MDSC and TAMs. This is a powerful tool for targeting M-MDSC and TAMs through FRβ. IL-6 has been previously demonstrated to be necessary for both M-MDSC and TAM development. However, total blockade of IL-6 impacts inflammatory components of the immune system. Targeting IL-6 through FRβ would provide a means to block M-MDSC and TAM in a manner that spares the rest of the immune component. Additionally, RNAseq analysis demonstrated a possible differential role for M-MDSC and TAMs at the tumor site despite similarities in suppressive function. FRβ+ M-MDSC had an expression profile that fit with a phenotype of immunosuppression and cross-talk ability to promote an anti-inflammatory environment while FRβ+ TAMs appeared well equipped to promote metastasis through expression of MMPs and markers of vascularization. The use of FRβ as a marker of functional M-MDSC and TAMs allows for a focused approach to identifying how these cells promote a pro-tumor environment. Additionally, pathways identified as important to function can then be immediately targeted through FRβ by delivering folate conjugated drugs