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Abstract Treatment of solid tumors remains challenging and therapeutic strategies require continuous development. Tumor-infiltrating macrophages play a pivotal role in tumor dynamics. Here, we present a Macrophage-Drug Conjugate (MDC) platform technology that enables loading macrophages with ferritin-drug complexes. We first show that macrophages actively take up human heavy chain ferritin (HFt) in vitro via macrophage scavenger receptor 1 (MSR1). We further manifest that drug-loaded macrophages transfer ferritin to adjacent cancer cells through a process termed ‘TRAnsfer of Iron-binding protein’ (TRAIN). The TRAIN process requires direct cell-to-cell contact and an immune synapse-like structure. At last, MDCs with various anti-cancer drugs are formulated with their safety and anti-tumor efficacy validated in multiple syngeneic mice and orthotopic human tumor models via different routes of administration. Importantly, MDCs can be prepared in advance and used as thawed products, supporting their clinical applicability. This MDC approach thus represents a promising advancement in the therapeutic landscape for solid tumors.

Abstract Hemoglobin (Hb) performs its physiological function within the erythrocyte. Extracellular Hb has prooxidative and proinflammatory properties and is therefore sequestered by haptoglobin and bound by the CD163 receptor on macrophages. In the present study, we demonstrate a novel process of Hb uptake by macrophages independent of haptoglobin and CD163. Unexpectedly, macrophages do not degrade the entire Hb, but instead transfer it to neighboring cells. We have shown that the phenomenon of Hb transfer from macrophages to other cells is mainly mediated by extracellular vesicles. In contrast to the canonical Hb degradation pathway by macrophages, Hb transfer has not been reported before. In addition, we have used the process of Hb transfer in anticancer therapy, where macrophages are loaded with a Hb-anticancer drug conjugate and act as cellular drug carriers. Both mouse and human macrophages loaded with Hb-monomethyl auristatin E (MMAE) effectively killed cancer cells when co-cultured in vitro.

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295-375 nm, fluorescence excitation.

Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability.