Cancer cell dependence on activated oncogenes is therapeutically targeted, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs and prevented or even reversed the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, which are responsible for loss of responsiveness to oncogene-targeted therapies.

Tumor-associated macrophages (TAM) are by now established as important regulators of tumor progression by impacting on tumor immunity, angiogenesis, and metastasis. Hence, a multitude of approaches are currently pursued to intervene with TAM's protumor activities, the most advanced of which being a blockade of macrophage-colony stimulating factor (M-CSF)/M-CSF receptor (M-CSFR) signaling. M-CSFR signaling largely impacts on the differentiation of macrophages, including TAM, and hence strongly influences the numbers of these cells in tumors. However, a repolarization of TAM toward a more antitumor phenotype may be more elegant and may yield stronger effects on tumor growth. In this respect, several aspects of TAM behavior could be altered, such as their intratumoral localization, metabolism and regulatory pathways. Intervention strategies could include the use of small molecules but also new generations of biologicals which may complement the current success of immune checkpoint blockers. This review highlights current work on the search for new therapeutic targets in TAM.

Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-IIhigh/trophic MHC-IIlow macrophage ratio was increased in tumors that produce anti-NRP-1 Nbs. This finding was corroborated by an increase in the expression of genes associated with MHC-IIhigh macrophages and a decrease in the expression of MHC-IIlow macrophage-associated genes in the macrophage pool sorted from anti-NRP-1 Nb-producing tumors. Moreover, we observed a significantly higher percentage of tumor-associated antigen-specific CD8+ T cells in tumors producing anti-NRP-1 Nbs. These data demonstrate that an intratumoral expression of NRP-1/Sema3A blocking biologicals increases anti-tumor immunity.

The tumor stroma has long been ignored as therapeutic target, but it has become clear that several stromal cell types play a nonredundant role during tumor progression. In particular, macrophages possess the capacity to stimulate tumor growth and metastasis via multiple mechanisms. [3]). Only upon experimentally re-educating TAMs, through the blockade of phagocytosis-inhibiting molecules such as CD47 [4], the provision of cytotoxicity-stimulating triggers such as CD40 and/or TLR ligands [5], the provision of macrophage repolarizing molecules such as histidine-rich glycoprotein [6], or the prevention of TAM migration to hypoxic areas [7], do these cells acquire antitumoral activity. In this respect, distinct TAM subpopulations exist within the same tumor-as demonstrated by earlier studies [8][9][10] and by Tymoszuk et al. in this issue of the European Journal of Immunology [11]-which might perform distinct functions. Hence, the ablation of tumor-promoting cells while leaving antitumoral populations unharmed could be envisaged as an anticancer strategy. Alternatively, preventing the establishment of the TAM pool could be another valid therapeutic strategy, but requires a better insight into the mechanisms governing the TAM pool size. Monocyte recruitment to the tissue and their differentiation into macrophages has long been known to supply sufficient amounts of phagocytes during inflammation, but more recently the importance of local macrophage proliferation at the site of inflammation has been underscored (reviewed in [12]). Tymoszuk et al.[11] demonstrate the contribution of both phenomena to TAM populations in the MMTV-Neu mouse model of oncogene-driven mammary carcinogenesis (Fig. 1) promoter contains four putative STAT1-binding GAS (interferongamma-activated sequences) elements, of which at least GAS1 binds STAT1 in response to IFN-γ treatment of cancer cells [11]. Importantly, the GAS1 sequence is conserved across mammalian species, including humans. CSF-1 drives hematopoietic stem cells toward the myeloid lineage and remains an important growth factor for the generation of monocytes and macrophages [21]. In reorganizing tissues-as a consequence of inflammation, wound healing or nonpathological events such as pregnancy-CSF-1 has been reported to regulate monocyte and macrophage dynamics. For example, in the myometrium of the pregnant uterus, CSF-1 regulates the macrophage pool by strongly increasing the extravasation of Ly6C hi monocytes and boosting local macrophage prolifera- Overall, the new information reported in the study by Tymoszuk et al. [11] provides further evidence for the existence of at least two main TAM subsets in tumors and identifies monocyte attraction and in situ macrophage proliferation as parallel mechanisms determining the TAM pool size. These data imply that antiproliferative agents currently in use as cancer therapeutics not only target cancer cells but might also hamper TAM functionality. CSF-1R signaling is instrumental in this scenario and constitutes a valid therapeutic target. ...