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The prevalence of liver cancer is constantly rising, with increasing incidence and mortality in Europe and the USA in recent decades. Among the different subtypes of liver cancers, hepatocellular carcinoma (HCC) is the most commonly diagnosed liver cancer. Besides advances in diagnosis and promising results of pre-clinical studies, HCC remains a highly lethal disease. In many cases, HCC is an effect of chronic liver inflammation, which leads to the formation of a complex tumor microenvironment (TME) composed of immune and stromal cells. The TME of HCC patients is a challenge for therapies, as it is involved in metastasis and the development of resistance. However, given that the TME is an intricate system of immune and stromal cells interacting with cancer cells, new immune-based therapies are being developed to target the TME of HCC. Therefore, understanding the complexity of the TME in HCC will provide new possibilities to design novel and more effective immunotherapeutics and combinatorial therapies to overcome resistance to treatment. In this review, we describe the role of inflammation during the development and progression of HCC by focusing on TME. We also describe the most recent therapeutic advances for HCC and possible combinatorial treatment options.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

It was previously reported that the activation of antitumor immune response by photodynamic therapy (PDT) is crucial for its therapeutic outcome. Excessive PDT-mediated inflammation is accompanied by immunosuppressive mechanisms that protect tissues from destruction. Thus, the final effect of PDT strongly depends on the balance between the activation of an adoptive arm of immune response and a range of activated immunosuppressive mechanisms. Here, with flow cytometry and functional tests, we evaluate the immunosuppressive activity of tumor-associated myeloid cells after PDT. We investigate the antitumor potential of PDT combined with indoleamine 2,3-dioxygenase 1 (IDO) inhibitor in the murine 4T1 and E0771 orthotopic breast cancer models. We found that the expression of IDO, elevated after PDT, affects the polarization of T regulatory cells and influences the innate immune response. Our results indicate that, depending on a therapeutic scheme, overcoming IDO-induced immunosuppressive mechanisms after PDT can be beneficial or can lead to a systemic toxic reaction. The inhibition of IDO, shortly after PDT, activates IL-6-dependent toxic reactions that can be diminished by the use of anti-IL-6 antibodies. Our results emphasize that deeper investigation of the physiological role of IDO, an attractive target for immunotherapies of cancer, is of great importance. Electronic supplementary material The online version of this article (10.1007/s00262-020-02528-5) contains supplementary material, which is available to authorized users.

Simple Summary Tumor-Associated Macrophages (TAMs) play an important role in the development of tumors, modulation of neoangiogenesis, immune suppression, and metastasis. High infiltration of macrophages in the tumor is also correlated with poor prognosis in several cancer types. Therefore, they became an attractive target for cancer immunotherapies. In this review, we describe the role of macrophages in tumorigenesis and summarize the most recent advances in the therapies targeting TAMs. Abstract Macrophages are critical mediators of tissue homeostasis and influence various aspects of immunity. Tumor-associated macrophages are one of the main cellular components of the tumor microenvironment. Depending on their activation status, macrophages can exert a dual influence on tumorigenesis by either antagonizing the cytotoxic activity of immune cells or, less frequently, by enhancing antitumor responses. In most situations, TAMs suppress T cell recruitment and function or regulate other aspects of tumor immunity. The importance of TAMs targeting in cancer therapy is derived from the strong association between the high infiltration of TAMs in the tumor tissue with poor patient prognosis. Several macrophage-targeting approaches in anticancer therapy are developed, including TAM depletion, inhibition of new TAM differentiation, or re-education of TAM activation for cancer cell phagocytosis. In this review, we will describe the role of TAMs in tumor development, including such aspects as protumorigenic inflammation, immune suppression, neoangiogenesis, and enhancement of tissue invasion and distant metastasis. Furthermore, we will discuss therapeutic approaches that aim to deplete TAMs or, on the contrary, re-educate TAMs for cancer cell phagocytosis and antitumor immunity.

Unsatisfactory response of tumors to chemotherapy is mainly related to impaired diffusion of the anticancer drug because of decreased drug uptake due to poor vasculature. Moreover, the drug is not able to penetrate the most hypoxic sites. Cells from these “untreated” sites are responsible for relapse and metastasis. However, these avascular regions attract macrophages that migrate even to areas far away from blood vessels. Therefore, they might constitute a unique delivery system of drug-containing particles to these parts of the tumor mass. A promising example of such particles that could be used is ferritins, whose caged architecture allows for efficient drug encapsulation and whose uptake from macrophage cells has been well demonstrated. Macrophages are also able to specifically and actively transfer these taken-up ferritins (loaded with the anticancer drugs) to cancer cells. This is the new mechanism discovered by our team and named TRAIN (TRAnsfer of Iron-binding proteiN). Thus, there is a possibility to use macrophages to deliver ferritin-encapsulated compounds directly to the tumor cells (MDC technology—Macrophage-Drug Conjugate). This is a completely new and modern approach to anticancer therapy and drug delivery. As such, we expect to be able to precisely administer drugs to the tumor site (even to the hypoxic regions), decreasing side effects of anticancer therapy. Acknowledgments: This research is founded by the European Research Council, Starting Grant McHAP: 715048. The dissemination of the project is founded by the KNOW (Leading National Research Centre), Scientific Consortium “Healthy Animal-Safe Food,” decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015. Citation Format: Bartłomiej Taciak, Maciej Białasek, Paulina Kucharzewska-Siembieda, Łukasz Kiraga, Aleksandra Szulc, Emilia Górka, Małgorzata Kubiak, Zofia Pilch, Katarzyna Tonecka, Zuzanna Sas, Agata Braniewska, Łukasz Cheda, Zbigniew Rogulski, Michał Godlewski, Alberto Boffi, Tomasz Rygiel, Magdalena Król. The macrophage-drug conjugate (MDC) as a “Trojan horse” approach in cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B24.

Tumor-infiltrating immune cells can impact tumor growth and progression. The inhibitory CD200 receptor (CD200R) suppresses the activation of myeloid cells and lack of this pathway results in a reduction of tumor growth, conversely a tumorigenic effect of CD200R triggering was also described. Here we investigated the role of CD200R activation in syngeneic mouse tumor models. We showed that agonistic CD200R antibody reached tumors, but had no significant impact on tumor growth and minor effect on infiltration of immune myeloid cells. These effects were reproduced using two different anti-CD200R clones. In contrast, we showed that CD200-deficiency did decrease melanoma tumor burden. The presence of either endogenous or tumor-expressed CD200 restored the growth of metastatic melanoma foci. On the basis of these findings, we conclude that blockade of the endogenous ligand CD200 prevented the tumorigenic effect of CD200R-expressing myeloid cells in the tumor microenvironment, whereas agonistic anti-CD200R has no effect on tumor development.

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.

Guitart et al. performed an in vivo genetic dissection of the Krebs cycle enzyme fumarate hydratase (Fh1) in the hematopoietic system. Their investigations revealed multifaceted functions of Fh1 in the regulation of hematopoietic stem cell biology and leukemic transformation., Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes.

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes.

Stimulation of Toll-like receptor 7 (TLR7) activates myeloid cells and boosts the immune response. Previously, we have shown that stimulation of the inhibitory CD200 receptor (CD200R) suppresses TLR7 signaling and that the absence of CD200R signaling leads to a decreased number of papillomas in mice. Here, we investigated the effects of agonistic anti-CD200R on the antitumor activity of a TLR7 agonist (R848) in a syngeneic mouse tumor model. Intratumoral administration of R848 inhibited the growth of the CT26 colon carcinoma and simultaneously decreased CD200R expression in tumor-infiltrating immune cells. The antitumor effects of R848 were potentiated by anti-CD200R. Successfully treated mice were resistant to rechallenge with the same tumor cells. However, the immediate antitumor effects were independent of lymphocytes, because treatment efficacy was similar in wild-type and Rag1tm1Mom mice. Administration of R848, particularly in combination with anti-CD200R, changed the phenotype of intratumoral myeloid cells. The infiltration with immature MHC-II+ macrophages decreased and in parallel monocytes and immature MHC-II− macrophages increased. Combined treatment decreased the expression of the macrophage markers F4/80, CD206, CD86, CD115, and the ability to produce IL1β, suggesting a shift in the composition of intratumor myeloid cells. Adoptively transferred CD11b+ myeloid cells, isolated from the tumors of mice treated with R848 and anti-CD200R, inhibited tumor outgrowth in recipient mice. We conclude that administration of agonistic anti-CD200R improves the antitumor effects of TLR7 signaling and changes the local tumor microenvironment, which becomes less supportive of tumor progression. Cancer Immunol Res; 6(8); 930–40. ©2018 AACR.