Multi-spectral immunofluorescence evaluation of the myeloid, T cell, and natural killer cell tumor immune microenvironment in chordoma may guide immunotherapeutic strategies

Abstract

BackgroundChordoma is a rare, invasive, and devastating bone malignancy of residual notochord tissue that arises at the skull base, sacrum, or spine. In order to maximize immunotherapeutic approaches as a potential treatment strategy in chordoma it is important to fully characterize the tumor immune microenvironment (TIME). Multispectral immunofluorescence (MIF) allows for comprehensive evaluation of tumor compartments, molecular co-expression, and immune cell spatial relationships. Here we implement MIF to define the myeloid, T cell, and natural killer (NK) cell compartments in an effort to guide rational design of immunotherapeutic strategies for chordoma.MethodsChordoma tumor tissue from 57 patients was evaluated using MIF. Three panels were validated to assess myeloid cell, T cell, and NK cell populations. Slides were stained using an automated system and HALO software objective analysis was utilized for quantitative immune cell density and spatial comparisons between tumor and stroma compartments.ResultsChordoma TIME analysis revealed macrophage infiltration of the tumor parenchyma at a significantly higher density than stroma. In contrast, helper T cells, cytotoxic T cells, and T regulatory cells were significantly more abundant in stroma versus tumor. T cell compartment infiltration more commonly demonstrated a tumor parenchymal exclusion pattern, most markedly among cytotoxic T cells. NK cells were sparsely found within the chordoma TIME and few were in an activated state. No immune composition differences were seen in chordomas originating from diverse anatomic sites or between those resected at primary versus advanced disease stage.ConclusionThis is the first comprehensive evaluation of the chordoma TIME including myeloid, T cell, and NK cell appraisal using MIF. Our findings demonstrate that myeloid cells significantly infiltrate chordoma tumor parenchyma while T cells tend to be tumor parenchymal excluded with high stromal infiltration. On average, myeloid cells are found nearer to target tumor cells than T cells, potentially resulting in restriction of T effector cell function. This study suggests that future immunotherapy combinations for chordoma should be aimed at decreasing myeloid cell suppressive function while enhancing cytotoxic T cell and NK cell killing.

Multimodal profiling of chordoma immunity reveals distinct immune contextures

“…Similar to our findings, the majority of these immune cells was confined to the stroma, although both cell types could be found in the tumour nodules 6,7,28 . Moreover, other lymphoid cells such as NK cells (or innate lymphoid cells) and B cells were considered rare in chordomas 7,23,28 . Using imaging mass cytometry, we expanded on the previously identified immune cell populations and were able to establish immunologically relevant interactions within the chordoma microenvironment.…”

“…These studies identified myeloid cells and T cells as the most predominant immune cell populations in chordomas 7,23,27,28 . Similar to our findings, the majority of these immune cells was confined to the stroma, although both cell types could be found in the tumour nodules 6,7,28 . Moreover, other lymphoid cells such as NK cells (or innate lymphoid cells) and B cells were considered rare in chordomas 7,23,28 .…”

“…Recently, several studies have contributed to the improved understanding of the chordoma microenvironment, based on approaches including immunohistochemistry, multispectral immunofluorescence or single cell-RNA sequencing 6,7,23,27,28 . These studies identified myeloid cells and T cells as the most predominant immune cell populations in chordomas 7,23,27,28 . Similar to our findings, the majority of these immune cells was confined to the stroma, although both cell types could be found in the tumour nodules 6,7,28 .…”

Chordoma recruits and polarizes tumor-associated macrophages via secreting CCL5 to promote malignant progression

“… 36–39 Surprisingly, single-cell RNA-sequencing and multiplex IF techniques were applied in studies of the chordoma TIME only since last year, 40 41 with their findings supporting the current results, as macrophages were reported to significantly infiltrate the chordoma parenchyma, while T cells exhibited stromal infiltration. 41 To our knowledge, this is the first report of spectral flow cytometry analysis in chordoma, also in combination with mIF for TIME characterization. Our findings suggest that immunotherapy approaches for chordoma should target the interaction of macrophage and chordoma cells or promote T-cell infiltration into the tumor parenchyma.…”

Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence

“…We hypothesized that all chordoma cell lines examined in vitro would preferentially express immunotherapeutic target surface ligands in a subpopulation of CSC marker-enriched cells. Multispectral immunofluorescence (mIF) is an imaging method by which we may individually identify cells of interest within the tumor microenvironment, detect protein coexpression on these cells, and understand the spatial relationship among these ( 26 ). We proposed that using mIF, we could detect the presence and relative location of CSCs within operative chordoma samples.…”