It is known that when primed with endotoxin, macrophages are more reactive and produce higher levels of inflammatory mediators upon subsequent stimulation, whereas endotoxin-tolerant macrophages have a blunted response and lower production of these mediators [1]. The effect of endotoxin-treated macrophages on cancer development has been explored [2,3], with interesting findings showing that endotoxin-tolerant macrophages create favourable conditions for tumor progression. It is much less known however, how such endotoxin-tolerant macrophages respond to contact with cancer cells.

One of the key effects of endotoxin tolerance is a shift in macrophage polarization. This process involves macrophages adapting to different functional states in response to environmental signals, including pathogen-associated molecular patterns (PAMPs) like bacterial endotoxins [2]. These states are broadly categorized into two main phenotypes: M1 (classically activated) [3,4] and M2 (alternatively activated) macrophages [5,6]. Each phenotype plays a distinct role in immune responses, inflammation and tissue homeostasis. Recognizing macrophage polarization is essential because it provides insight into the immune system's balance between pro-inflammatory and anti-inflammatory responses. This understanding is critical for developing targeted therapies for various conditions, including infections, chronic inflammatory diseases, and cancer.

To identify macrophage polarization, various markers and factors indicative of different macrophage phenotypes can be assessed. These include cytokines, enzymes, surface markers, and the production of reactive oxygen species (ROS). It is well known that interleukin (IL) 6, tumor necrosis factor (TNF) α and IL-1β are produced in high levels by M1 macrophages and are key indicators of the pro-inflammatory state [7]. They drive inflammation and help recruit other immune cells to sites of infection [8] or injury [9]. Enzymes such as inducible nitric oxide synthase (iNOS) [10,11] and cyclooxygenase-2 (COX-2) [12] are directly involved in the metabolic activities and responses of macrophages. For instance, iNOS catalyzes nitric oxide production, which plays a role in pathogen killing and inflammation [13], but can also contribute to tissue damage if overproduced, while COX-2 generates pro-inflammatory prostaglandins [14,15]. ROS, generated by various sources including NADPH oxidase, play a role in pathogen destruction and tissue damage, further amplifying the inflammatory response [16,17]. These enzymatic activities provide insights into the functional roles of macrophages in inflammation and tissue repair that surface markers alone cannot reveal. In contrast to the M1, M2 macrophages are characterized by elevated expression of arginase-1 (Arg-1), mannose receptor (CD206), and the anti-inflammatory cytokine IL-10. These macrophages are primarily activated by cytokines and factors such as IL-4, IL-13, IL-10, immune complexes, hormones, or agonists of adenosine A2A receptors (A2AR). M2 macrophages play a crucial role in tissue repair, angiogenesis, and various metabolic processes, contributing to the resolution of inflammation and promoting healing [[18], [19], [20], [21], [22]].

Finally, surface markers, such as CD80 and CD163 complement cytokine and enzymatic markers by providing additional specificity for identifying M1 and M2 macrophages. CD80 marks the pro-inflammatory M1 state [23], while CD163 is a hallmark of the anti-inflammatory M2 state [24,25].

This study aims to determine whether endotoxin tolerance triggers a shift in macrophage polarization and metabolism, focusing on evaluating the response of endotoxin-tolerant macrophages upon contact with cancer cells in vitro.