Natural killer cells (NK cells) are a crucial constituent of the innate immune system as they mediate immunity against viruses, bacteria, parasites, and most importantly, tumor cells. The exact mechanism of how the innate immune system and specifically NK cells interact with cancer cells is complex and is yet to be understood. Several factors that constitute the tumor microenvironment (TME) such as hypoxia and TGF-β are believed to play a role in the complex physiological reaction of NK cells to tumor cells. On the other hand, several risk factors are implicated in the development and progression of breast cancer, most importantly: obesity. Cytokines released from adipose tissue such as adipokines, leptin, and resistin, among others, are also believed to facilitate tumor progression. In this study, we aimed to build a triad of breast cancer, obesity, and NK cell dysfunction to elucidate a link between these pillars on a cellular level. Directing efforts towards solidifying the link between these factors will help in designing a targeted immunotherapy with a low side-effect profile that can revolutionize breast cancer treatment and improve survival in obese patients.

Background and aim Breast cancer (BC) is the second most common global cause of cancer deaths among women. Several immune cells are identified in the tumor microenvironment of BC patients, including tumor‐associated macrophages. We aimed at exploring the expression of distinct functional phenotypes using macrophages’ markers, where CD68 is a pan‐macrophage marker, CD86 is a marker expressed in polarized M1 subtype, and CD163 is expressed in M2 polarized subtype. Methods A retrospective study was performed using 90 formalin‐fixed paraffin‐embedded BC specimens for the immunohistochemical analysis of CD68, CD86 and CD163. Also, an in silico tool, UALCAN, was used on a larger cohort (n=1,081) of BC patients to investigate the expression of these markers. The macrophages’ markers were then associated with the clinicopathological parameters of BC patients. Triple‐negative BC cell line “CAL‐51” and luminal BC cell line “MCF‐7” were used to collect their respective supernatants that were added to THP‐1 derived macrophages. Then CD86 and CD163 were assessed using western blot. Results The pan‐marker of macrophages, CD68, along with the M1 CD86 marker and M2 CD163 marker, showed positive results in all the 90 investigated patients (Figure 1). CD86 expression was significantly associated with body mass index (BMI) and Ki‐67 proliferation marker. On the other hand, CD163 was significantly correlated with tumor size, estrogen, progesterone receptors, and BC molecular subtypes. Moreover, the high expression of CD86 and CD163 showed unfavorable outcome and survival of BC patients. In silicoanalysis revealed a significant increase in the CD86 expression in BC patients, especially in the triple‐negative subtype. Similarly, CD163 expression was found to be higher in the triple‐negative subgroup compared to the luminal group (Figure 2A). Additionally, in vitro work showed that the macrophages induced from the monocytic THP‐1 cell line express high levels of CD163 upon the exposure to conditioned media collected from the luminal BC cell line “MCF‐7”, thus showing M2 phenotype. On the contrary, CD163 expression was reduced upon the exposure to the conditioned media collected from the triple‐negative BC cell line “CAL51”, indicating more M1 phenotype (Figure 2B). Conclusion Tumor‐associated macrophages are associated with cancer progression and molecular subtypes. Hence, identifying the macrophages polarization profiles in each molecular subtype might aid their use as potential therapeutic targets.