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. 2022 Oct 21:12:1012058.
doi: 10.3389/fonc.2022.1012058. eCollection 2022.

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

Diana C Lopez  1   2 , Yvette L Robbins  3 , Joshua T Kowalczyk  4 , Wiem Lassoued  4 , James L Gulley  4 , Markku M Miettinen  5 , Gary L Gallia  6   7 , Clint T Allen  3 , James W Hodge  4 , Nyall R London Jr  1   6   7
Affiliations

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

Diana C Lopez et al. Front Oncol. .

Abstract

Background: Chordoma 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.

Methods: Chordoma 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.

Results: Chordoma 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.

Conclusion: This 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.

Keywords: T cells; chordoma; immunotherapy; myeloid cells; natural killer cells; tumor immune microenvironment (TIME).

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Conflict of interest statement

NL receives research funding from Merck Sharp & Dohme, LLC regarding HPV related sinonasal carcinomas, holds stock in Navigen Pharmaceuticals and was a consultant for Cooltech Inc., none of which are relevant to the present manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Myeloid cells are found within the chordoma tumor immune microenvironment. (A) Representative photomicrographs from one chordoma of merged and single-color immunofluorescence images assessing the presence of myeloid cells with a validated panel of six biomarkers, CD11b (yellow, Opal 570), HLA-DR (red, Opal 690), CD14 (turquoise, Opal 480), CD15 (orange, Opal 620), CD68 (green, Opal 520), and CK (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. High expression of CD68 identified pan macrophages. Co-localization of CD11b and CD14 without CD15 and HLA-DR identified monocytes. Co-localization of CD11b and CD15 without CD14 and HLA-DR identified PMNs. Expression of CK identified chordoma tumor cells. (B) Quantification of pan macrophages, monocytes, and PMNs per mm2 in chordoma tumor and stroma (n = 56 individual tumors). Wilcoxon signed rank tests with p value threshold of <0.05 were used to compare tumor and stroma myeloid cell infiltrate. *p ≤ 0.05, **p ≤ 0.01, ns, non significant. CK, cytokeratin; DAPI, 4′,6-diamidino-2- phenylindole; PMN, polymorphonuclear cells.
Figure 2
Figure 2
T cells are found in abundance within the chordoma tumor immune microenvironment, most often within the stroma. (A) Representative photomicrographs from one chordoma of merged and single-color immunofluorescence images assessing the presence of T cells with a validated panel of six biomarkers, CD8 (yellow, Opal 570), CD4 (green, Opal 520), FOXP3 (turquoise, Opal 480), Ki67 (red, Opal 690), PD1 (orange, Opal 620), and CK (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Expression of CD4 without CD8 and FOXP3 identified CD4+ T helper cells, while expression of CD8 without CD4 and FOXP3 identified CD8+ cytotoxic effector T cells. Co-localization of CD4 and FOXP3 without CD8 identified Tregs. T cells with positive Ki67 nuclear expression were proliferating, and PD1 positivity denoted PD1+ T cells. Expression of CK identified chordoma tumor cells. (B) Quantification of CD4+ T cell, CD8+ T cell, and Treg cell density (per mm2), as well as proliferating and PD-1+ subcategories of these are compared between chordoma tumor and stroma (n = 57 individual tumors). Wilcoxon signed rank tests with p value threshold of <0.05 were used to compare tumor and stroma T cell infiltrate. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns, non significant. CK, cytokeratin; DAPI, 4′,6-diamidino-2- phenylindole; PD1, programmed cell death protein 1.
Figure 3
Figure 3
Chordoma tumor parenchymal T cell inclusion versus exclusion. T cell compartment inflammation in chordoma more commonly takes on a tumor parenchymal exclusion, rather than inclusion, pattern (n = 57 individual tumors). (A) A tumor:stroma T cell infiltrate ratio >1.5 defined substantial tumor parenchymal inclusion. A stroma:tumor T cell infiltrate ratio >4.5 defined substantial tumor parenchymal exclusion. (B) Representative chordoma samples with heavy tumoral infiltrate (left) versus heavy stromal infiltrate (right).
Figure 4
Figure 4
NK cells are sparsely found within the chordoma tumor immune microenvironment. (A) Representative photomicrographs from one chordoma of merged and single-color immunofluorescence images assessing the presence of NK cells with a validated panel of five biomarkers, CD16 (orange, Opal 620), CD56 (red, Opal 690), CD3 (turquoise, Opal 480), Granzyme B (green, Opal 520), and CK (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Co-localization of CD16 and CD56 without CD3 and a decrease size threshold compared to tumor cells identified NK cells. Expression of Granzyme B identified Granzyme B+ or Granzyme B- NK cells. Expression of CK identified chordoma tumor cells. (B) Quantification of NK cells overall, Granzyme B+, and Granzyme B- NK cells per mm2 in chordoma tumor and stroma (n = 54 individual tumors). Wilcoxon signed rank tests with p value threshold of <0.05 were used to compare tumor and stroma NK cell infiltrate. ns, non significant. NK cell, natural killer cell; CK, cytokeratin; DAPI, 4′,6-diamidino-2- phenylindole.
Figure 5
Figure 5
HALO® spatial proximity analyses of immune cells to chordoma cells demonstrate that T cells are nearer than myeloid cells to tumor cells. (A) Sacral chordoma specimen with three categories of T cells, CD4+ T cells, CD8+ T cells, and Tregs shown in blue, yellow, and red respectively. Tumor cells are shown in green. A segment of heavy T cell infiltrate is magnified. (B) Real time proximity analysis between CD4+ T cells (green, Opal 520) and chordoma cells (white, Opal 780) with bars measuring distance between these superimposed on the image in white. (C) Proximity line series between T cells and chordoma tumor cells. (D) Proximity analysis histograms of myeloid cells (top row, red) and T cells (bottom row, blue) to chordoma tumor cells within a 100µm radius by progressive segments of 20µm bands (n = 56 individual tumors).
Figure 6
Figure 6
Spatial characterization of distance between Tregs to proliferating and nonproliferating CD8+ T cells in chordoma tumor slides (n = 57). Percentage of Tregs within 20µm of nonproliferating CD8+ T cells (mean 8.90 ± SEM 1.74) is significantly greater than that within 20µm of proliferating CD8+ T cells (mean 1.34 ± SEM 0.57) (p < 0.0001), shown above. A Wilcoxon signed rank test with a p value threshold of <0.05 was used. ****p ≤ 0.0001. Proximity analysis histograms of Tregs within a 20µm radius of nonproliferating CD8+ T cells (left, blue) and proliferating CD8+ T cells (right, red) by progressive segments of 1µm bands, shown below.
Figure 7
Figure 7
Chordoma myeloid, T cell, and NK cell density does not significantly differ by anatomic site. Immune infiltrate differences among chordomas (n = 20) originating from skull base (n = 9), spine (n = 3), or sacrum/coccyx (n = 8) were assessed with two-way ANOVA analysis using a p value threshold of <0.05. Chordoma myeloid, T cell, and NK cell density does not significantly differ by disease stage. Immune infiltrate differences between chordoma patients (n = 20) with primary and advanced disease were assessed with Mann Whitney tests using a p value threshold of <0.05. Advanced disease was defined as including cases of locally progressive and locally advanced, destructive disease. ns, non significant. NK cell, natural killer cell.
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