The Immunological Landscape of M1 and M2 Macrophages and Their Spatial Distribution in Patients with Malignant Pleural Mesothelioma

Legend of Supplementary Figures 

Supplementary Figure 1. Schematic of the action points associated with tumor-associated macrophages targeted by immunotherapy investigated in clinical trials. The schematic represents the most relevant pathways targeted by drugs investigated in clinical trials. Note the wide range of agents with action on different cell components, from targets such as immune checkpoint inhibitors to targets of direct action on DNA transcription, such as action on histones. The images were generated using BioRender (https://www.biorender.com/).

Supplementary Figure 2. Representative examples of unmixed and mixed multispectral images from controls of human reactive tonsil and tuberculous pneumonitis. Unmixed images (20× magnification; scale bars represent 50 µm on each image) showing the individual marker’s expression plus DAPI (4′,6-diamidino-2-phenylindole) and their composite mixed image contained all the markers plus DAPI from human reactive tonsil and acute tuberculous pneumonia: cytokeratin (CK), CD68, CD206, CD86, CD163, Arg-1, MRP8-14, and PD-L1. The images were generated using the PhenoImager 1.0.13 scanner system and inForm 2.4.8 image analysis software (Akoya Biosciences).

Supplementary Figure 3. Tumor gene expression according to sex. Boxplots showing a significant correlation between high expression of transcripts per million of CD274, IL12B, and INHBA and male patients (P=3.79E-04, P=1.40E-03, and P=5.56E-03, respectively).  Data from 87 samples was used. Boxplots show the median (bar), interquartile range (Q2 and Q3), and maximum and minimal values. The data were downloaded from UALCAN, from the expression profiles for each gene analyzed. Expression and group status were compared by the platform itself using Student t-test. *P<0.05; **P<0.01.

Supplementary Figure 4. Tumor gene expression according to histotype. Boxplots showing high expression of IL1B and INHBA in the biphasic histotype compared to epithelioid (P=2.55E-02, P=4.79E-02, respectively), while epithelioid showed upregulation of CHI3L1 compared to biphasic (P=1.62E-02). Furthermore, IL6 showed high expression in the biphasic histotype compared to diffuse malignant (P=2.99E-02). Lastly, CHI3L1 showed high expression in diffuse malignant compared to biphasic (P=6.3E-03). Data from 87 samples were used. Boxplots show the median (bar), – interquartile range values (Q2 and Q3), and maximum and minimal values. The data were downloaded from UALCAN, from the expression profiles for each gene analyzed. Expression and group status were compared by the platform itself using Student t-test. *P<0.05; **P<0.01.

Supplementary Figure 5. Tumor gene expression according to clinical stage. Boxplots showing significant overexpression of CD163 and MRC1 in stage IV compared to stage II (P=4.42E-02, P=2.32E-02, respectively), and overexpression of IL6 in stage III compared to stage II (P=2.65E-02). Data from 87 samples were used. Boxplots show the median (bar), interquartile range values (Q2 and Q3), and maximum and minimal values. The data were downloaded from UALCAN, from the expression profiles for each gene analyzed. Expression and group status were compared by the platform itself using Student t-test. *P<0.05.

Supplementary Figure 6. Tumor gene expression according to nodal metastasis status. Boxplots showing significant overexpression of mRNA of CD274 in the N3 category compared to N0 (P=2.91E-02) and of CHI3L1 in N0 compared to N1 (P=2.61E-02), N2 compared to N1 (P=4.46E-02) and N3 compared to N2 (P=4.84E-02). FN1 mRNA overexpression was significantly associated with N3 compared to N0 (P=2.23E-04), N1 (P=8.57E-04), and N2 (P=1.22E-04). Data from 87 samples were used. Boxplots show the median (bar), interquartile range values (Q2 and Q3), and maximum and minimal values. The data were downloaded from UALCAN, from the expression profiles for each gene analyzed. Expression and group status were compared by the platform itself using Student t-test. *P<0.05; **P<0.01.

Supplementary Figure 7. Prognostic curve of hub genes. The prognostic significance of the 18 TAM genes involved in TME signaling pathways from patients with MPM. Kaplan-Meier survival curves show the association between the TAM genes and OS. Red lines indicate high expression (>3rd quartile) and blue lines indicate low/medium expression (≤3rd quartile). Significant correlations were seen between high expression of NOS2 and poor OS (P=0.049), high expression of INHBA with poor OS (P<0.0001), and high expression of FN1 and poor OS (P<0.0001). Data from 87 samples were used. Kaplan‐Meier curves were downloaded from UALCAN, from the expression profiles for each of the genes analyzed. The significance of survival impact was measured by log-rank test, and P-values were provided by the UALCAN platform.