Identification of immunosuppressive factors in retinoblastoma cell secretomes and aqueous humor from patients

Abstract

The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor‐associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163+ protumoral M2‐like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2‐like polarization. M2‐like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8+ tumor‐infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD‐L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3+ Tregs infiltrating tumors. We confirmed the pathology results using single‐cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single‐cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2‐like polarization upon exposure to retinoblastoma‐conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.

Proteomics of Aqueous Humor as a Source of Disease Biomarkers in Retinoblastoma

“…Our finding of MIF to be highly expressed in active RB agrees with the results of a recent study showing that this protein, among others, is likely involved in the production of an immunosuppressive microenvironment in these intraocular tumors [ 23 ].…”

“…In recent years, several studies have reported that cell free DNA isolated from the AH of enucleated or conservatively treated eyes with RB can be used to characterize the genetics of the disease [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ], and it may also correlate with clinical outcomes [ 14 , 15 , 16 ]. On the other hand, the modulation of several proteins has been described in AH obtained after enucleation [ 17 , 18 , 19 , 20 , 21 , 22 ] or collected from patients who underwent intravitreal chemotherapy [ 23 ].…”

Immune Microenvironment in Childhood Cancers: Characteristics and Therapeutic Challenges

“…Tumor cells reprogram TAMs to promote immune suppression and resistance to anti-tumor drugs. This is supported by studies demonstrating that macrophages cultured in a conditioned medium obtained from retinoblastoma cells exhibit a more pro-tumor M2 phenotype [ 27 , 28 ]. M2-type TAMs are associated with various other types of pediatric tumors, including osteosarcoma, rhabdomyosarcoma, leukemia, lymphoma, and brain tumors.…”

“…Similarly, in retinoblastoma, the microenvironment is enriched with pro-tumorigenic macrophages expressing the M2-type microglial marker Iba1+ [ 27 ]. The population of these TAMs increases after therapy [ 27 ]. Tumor cells reprogram TAMs to promote immune suppression and resistance to anti-tumor drugs.…”

Follow‐up of intraocular retinoblastoma through the quantitative analysis of conserved nuclear DNA sequences in aqueous humor from patients

“…All cultures were under passage 20. As a quality control, we determined the short tandem repeat profiles of the primary cells, which we reported elsewhere [ 17 ].…”