Imaging MC and Data Session

Affiliation

Abstract

Biosketch

Charité – Universitätsmedizin Berlin, Department for Gastroenterology, Infectious Diseases and Rheumatology, Germany

Fatty acid metabolism-dependent polarization of tumor associated macrophages

Colorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer-related deaths worldwide. An interplay between tumor cells and surrounding stromal and immune cells characterizes solid tumors such as CRC. One prominent immune cell type is tumor-associated macrophages (TAMs), which typically display a pro-tumorigenic and anti-inflammatory phenotype. Previous studies have shown a negative correlation between overall survival and the abundance of specific TAM subsets in CRC. Our group recently described a mechanistic link between the uptake and metabolism of fatty acids and the polarization of macrophages towards a pro-tumorigenic and immunosuppressive phenotype. To study the presence and metabolism of TAMs, we established a 36-plex imaging mass cytometry (IMC) panel covering immune cell, cancer-specific and metabolic markers on formalin-fixed and paraffin-embedded (FFPE) samples. Using this panel, we analyzed a cohort of CRC patients, including samples of healthy areas, tumor center and infiltrating margin, organized in tissue microarrays. Additionally, we applied a 7-plex immunofluorescence panel on consecutive tissue sections to study the presence of lipid droplets using ADRP, a structural protein of lipid droplets, as well as epithelial and major immune cell markers. By combining both methods, we were able to identify lipid droplet-bearing cells and correlate their presence to the deep immune phenotyping and the activation of metabolic pathways in various immune cell subsets. Subsequently, these cellular and metabolic features were correlated with clinical parameters such as prognosis. The understanding of fatty acid metabolism of tumor-infiltrating immune cells creates new possibilities for cancer-targeted therapeutic interventions.

University Medical Center Freiburg, Clinic for Internal Medicine II, Germany

Deep profiling of the tumor immune microenvironment for personalized treatment for biliary tract cancer

Background and Aims:

Biliary tract cancers (BTCs) represent a heterogeneous group of rare, aggressive malignancies with low survival rates and increasing incidence. 3 BTC patients that progressed under first-line chemotherapy were screened by next generation sequencing for tumor mutational profiling and determined to have homologous recombination deficiency due to mutations in BRCA and BAP1 and were thus treated with PARP inhibitors. This study sought to understand if analysis of the tumor immune microenvironment (TiME), may provide additional insights into the suitability of immunotherapy options. We thus performed IMC analysis before PARP treatment and determined the tumor-immune architecture.

Methods:
Tumor samples from three BTC patients subsequently treated with a PARP inhibitor targeting specific DNA damage pathway mutations and exhibiting distinct clinical outcomes, were selected for highly multiplexed imaging mass cytometry (IMC) (42 markers, 3 ROIs each from tumor, margin and adjacent regions of BTC resections).

Results:
Clinically, two out of three patients with BRCA mutations showed complete remission to PARP inhibitors, in contrast, the patient with BAP1 syndrome experienced significant tumor progression. IMC analysis revealed distinct marker expression patterns in tumor, margin, and healthy tissues, including CA19.9, TROP-2, and Claudin18.2 in tumor regions, with HePar expression absent. Immune cells were present in both tumor and stromal regions, with notable differences in immune cell populations. The BAP1-mutated sample exhibited high immune cell infiltration, including activated CD8 T cells (TRM and TEX subsets), along with Granulocytes, NK cells, CD4 memory T cells and B cells. In contrast, immunosuppressive populations such as M2-like tumor-associated macrophages, Tregs, and MDSCs were enriched in BRCA-mutated samples. We applied a spatial immune classification developed for primary liver cancer, indicating an enriched immunotype in the BAP1-mutated sample and a depleted immunotype in the BRCA-mutated sample. Subsequent treatment with anti-PD-1 and TKI therapy for the BAP1-mutation patient resulted in a partial response and longterm survival.

Conclusions:
The tumor immune architecture appears to be connected to different genetic drivers of homologous mismatch repair insufficiency in BTC. High infiltration with immune cells in the BAP1 patient was observed in the context of response to anti-PD-1 / TKI therapy. Spatial profiling of the tumor-immune architecture may synergize with mutational profiling for improved personalized decision making in BTC patients.

Université Paris-Saclay, Inserm, CEA, Center for Immunology of Viral, Auto-immune, Hematological
and Bacterial diseases (IMVA-HB/IDMIT), Fontenay-aux-Roses, France

Role of antigen bio-distribution and persistence in early and long-term immune responses to the yellow fever vaccine in non-human primates

Purpose:
Despite recent advances in vaccine development, gaps remains to understand the mechanisms driving long-term memory response to vaccines. The purpose of the work is to characterize the role of vaccine antigen persistence in generating durable immunity. Using the live-attenuated yellow fever (YF)-17D vaccine, one of the most efficient and durable vaccine, we explored molecular and cellular interactions in tissues of immunized non-human primates.
Methods:
Two groups of cynomolgus macaques were either vaccinated with a commercial dose of live-attenuated vaccine or with the same dose of a β-propiolactone-inactivated vaccine adjuvanted with alum. Animals also received a 5-Iodo-2’-deoxyuridine (IdU) treatment to follow the mobilization and persistence of immune cells following immunization. Longitudinal systemic immune responses were characterized by mass cytometry, as well as viral load by qPCR, and neutralizing antibodies. At necropsy, different types of tissues were collected at several timepoints to follow antigen persistence by immunohistofluorescence (IHF) and in situ hybridisation; and to explore tissue immune responses by imaging mass cytometry (IMC). Results were compared to data obtained from tissues of macaques infected wild type Asibi strain.
Results:
Low and transient viremia is observed in vaccinated animals (reaching 103 to 104 copies/mL) at approximately day 3 pi. Longitudinal analysis of immune responses by mass cytometry shows a high mobilization of innate and adaptive immune cells at early time-points. Adaptive T and B cells show persistent activation in the long term (up to 12 month pi) as demonstrated by the persistence of IdU+ cells. Neutralizing antibodies were shown to persist for the live-attenuated vaccine whereas they decreased significantly around 3 months pi for the inactivated vaccine. By comparing these strategies, we identified differences in regulatory T cells and three types of circulating follicular helper cells (CD127+ PD-1-, CD127mid PD-1- CD25+, and CD127lo PD-1+) that could affect antibody persistence. In the tissues, YF antigens were detected at early timepoints mainly in the liver, although at lower rates than the infected animals, and in the spleen.
Conclusion:
Our data enhance the understanding of the immunological mechanisms of YF-17D vaccination. Together, they highlight the relevance of specific immunological mechanisms that occur after vaccination.