Understanding the fate of slan+ monocytes in the tumor microenvironment

slan+monocytes represent a major subset (50-60%) of peripheral non-classical monocytes in humans. These cells express the slan antigen, a glycosilated modification of P-selectin glycoprotein ligand-1 (PSGL-1), and have been found in different tissues under inflammatory conditions (such as psoriasis, Crohn disease, etc.), including peripheral lymphoid and non-lymphoid organs. Immuno histochemistry (IHC) experiments made by our group pointed for a selective accumulation of slan+cells in metastatic-tumor draining lymph nodes (M-TDLNs) and in various types of non-Hodgkin lymphomas (NHL), especially in the diffuse large B cell lymphoma subset (DLBCL), but not in primary carcinomas from a variety of organs. In the former cases, slan+cells resembled either DC-like or macrophage-like cells. Subsequently, peripheral slan+monocytes have been found to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of the anti-CD20 monoclonal antibody, known as Rituximab (RTX), towards B cells from healthy donors (HDs), neoplastic B cells from patients, and B-cell lines. ADCC was performed by slan+monocytes at levels likely equivalent to those exerted by NK cells, which play an important role in tumor immuno surveillance, including B cell lymphoma. In another study, our group investigated the fate of slan+monocytes in human tonsils by comparing the gene expression profiles of tonsil-resident slan+cells with those of peripheral slan+monocytes from HDs. The findings revealed that tonsil slan+cells display a macrophage-like transcriptional signature, suggesting that peripheral slan+monocytes undergo a transition toward a macrophage-like phenotype upon infiltration into tonsil tissue. To validate the previously described IHC findings at the molecular level, I then compared the tonsil gene signature of slan+cells with publicly available single- cell RNA sequencing (scRNA- seq) datasets of human macrophages present in various primary tumors (i.e., breast cancer, colorectal carcinoma, etc) and M-TDLNs. To our surprise, these preliminary in silico transcriptomic analyses revealed that the tonsil gene signature of slan+ cells is clearly enriched in discrete macrophage populations across primary tumor datasets, other than M-TDLNs, thus contrasting with previous IHC evidence. Therefore, during my doctoral research, I focused on answering two main questions derived from the experimental observations described above: 1. To decipher the underlying effector mechanism(s) employed by slan+monocytes to mediate antibody-dependent tumor cell targeting and killing. 2. To clarify the discrepancy between the IHC and bioinformatic data concerning the presence of slan+c ells in primary tumors. To address issue 1), I performed blocking experiments, flow cytometry analysis, as well as confocal microscopy and live- cell imaging assays to ultimately uncover that slan+monocytes kill tumor cells via trogoptosis, a lytic (i. e. necrotic) type of cell death that is one of the possible consequences of trogocytsis, a process of cell-cell interaction during which an effect or cell nibbles, and then rapidly ingests, membrane fragments of a target c ell. To address issue 2), and given the limited availability of cancer samples, I chose to use in vitro experimental models. Accordingly, I isolated circulating CD14++ CD16-classical monocytes, as well as slan+/slan- non-classical monocytes, from the buffy coats of HDs and then extracted their total RNA for bulk RNA-sequencing analysis, either immediately after isolation from the blood or following 5 days of in vitro culture with lymphoma-derived conditioned medium (DCM). The latter condition promotes the maturation of each monocyte subset into its monocyte-derived macrophage cells, putatively mimicking those present in tumors. Finally, I assessed how the gene expression profiles of these in vitro- cultured cells matched with those of publicly available scRNA-seq data of tumor macrophages. Results from issue 1) exploited slan+monocyte-mediated trogoptosis as a novel weapon and final mechanism for ADCC, adopted by slan+monocytes to kill cancer cells. This could be therapeutically relevant, especially in those cases where NK cells are ineffective. Results from issue 2) indicated that DCM-treated different monocyte subsets undergo transcriptional repr ogramming, acquiring a gene expression signature that resembles that of tumor-associated macrophages. However, the reprogramming varies quantitatively among monocyte subsets. Whether these quantitative differences in the gene expression profiles reflect a possible divergent fate- of individual monocyte subsets within the tumor microenvironment remains to be established. Nonetheless, these findings underscore the need to identify new markers that can clearly distinguish the cells of origin of macrophages infiltrating tumor tissue.