Carolina Giordano

Craniopharyngiomas continue to present a challenge in clinical practice due to their heterogeneity and unpredictable adherence to vital neurovascular structures, particularly the hypothalamus. This results in different degrees of hypothalamus–pituitary axis dysfunction and a lack of uniform consensus and treatment guidelines regarding optimal management. MRI and CT are complementary techniques in the preoperative diagnostic phase, enabling the precise definition of craniopharyngioma size, shape, and consistency, as well as guiding classification into histopathological subtypes and topographical categories. Meanwhile, MRI plays a crucial role in the immediate postoperative period and follow-up stages by identifying treatment-related changes and residual tumors. This pictorial essay aims to provide an overview of the role of imaging in identifying variables indicative of the adherence degree to the hypothalamus, hypothalamic–pituitary dysfunction, the extent of surgical excision, and prognosis. For a more comprehensive assessment, we choose to distinguish the following two scenarios: (1) the initial diagnosis phase, where we primarily discuss the role of radiological variables predictive of adhesions to the surrounding neurovascular structures and axis dysfunction which may influence the choice of surgical resection; (2) the early post-treatment follow-up phase, where we discuss the interpretation of treatment-related changes that impact outcomes.

The primary treatment for glioblastoma (GBM) is removing the tumor mass as defined by MRI. However, MRI has limited diagnostic and predictive value. Tumor-associated macrophages (TAM) are abundant in GBM tumor microenvironment (TME) and are found in peripheral blood (PB). FKBP51 expression, with its canonical and spliced isoforms, is constitutive in immune cells and aberrant in GBM. Spliced FKBP51s supports M2 polarization. To find an immunologic signature that combined with MRI could advance in diagnosis, we immunophenotyped the macrophages of TME and PB from 37 patients with GBM using FKBP51s and classical M1-M2 markers. We also determined the tumor levels of FKBP51s, PD-L1, and HLA-DR. Tumors expressing FKBP51s showed an increase in various M2 phenotypes and regulatory T cells in PB, indicating immunosuppression. Tumors expressing FKBP51s also activated STAT3 and were associated with reduced survival. Correlative studies with MRI and tumor/macrophages cocultures allowed to interpret TAMs. Tumor volume correlated with M1 infiltration of TME. Cocultures with spheroids produced M1 polarization, suggesting that M1 macrophages may infiltrate alongside cancer stem cells. Cocultures of adherent cells developed the M2 phenotype CD163/FKBP51s expressing pSTAT6, a transcription factor enabling migration and invasion. In patients with recurrences, increased counts of CD163/FKBP51s monocyte/macrophages in PB correlated with callosal infiltration and were accompanied by a concomitant decrease in TME-infiltrating M1 macrophages. PB PD-L1/FKBP51s connoted necrotic tumors. In conclusion, FKBP51s identifies a GBM subtype that significantly impairs the immune system. Moreover, FKBP51s marks PB macrophages associated with MRI features of glioma malignancy that can aid in patient monitoring.Significance:Our research suggests that by combining imaging with analysis of monocyte/macrophage subsets in patients with GBM, we can enhance our understanding of the disease and assist in its treatment. We discovered a similarity in the macrophage composition between the TME and PB, and through association with imaging, we could interpret macrophages. In addition, we identified a predictive biomarker that drew more attention to immune suppression of patients with GBM.

IB of the expression levels of FKBP51s in 7 GB tumor cell lysates with the respective densitometric analysis of IB. Bands were quantitated by densitometry, using ImageJ 1.42q for Macintosh. Integrated ODs were normalized to two relative housekeeping genes (G3PDH and Tubulin).

Necrosis score in relation to other MRI features: Pearson r coefficient and p values are indicated for each variable. Correlation between necrosis score and ITSS.

Effect of the tumor on macrophage polarization. Cocultures of M0 cells with GB138 (A) and GB83 (B), either adherent cell or spheroid. Expression of CD163 on macrophages was upregulated in cocultures with adherent cells (red column) more than spheroids (gray column). Part of CD163 macrophages expressed FKBP51s (N = 3). Cocultures of M0 cells with GB138 (C) and GB83 (D), either adherent cell or spheroid. Expression of HLA-DR (MFI) on macrophages was upregulated in cocultures with spheroids (gray column). HLA-DR's expression was also registered on tumor cells, particularly spheroids (N = 3). E, Immunoblot of pSTAT6 expression in PBMCs of 5 patients with GBM. Samples 2 and 3 show pSTAT6. Corresponding values of CD163/FKBP51s (top) and PD-L1/FKBP51s or CD206/FKBP51s (bottom) PB-TAMs are also shown. F, Graphic representation of pSTAT6 levels (MFI) in CD163 macrophages from cocultures (N = 5). Coexpression of FKBP51s in CD163 significantly increased pSTAT6 (see Supplementary Fig. S5F and S5G). G, Immunoblot of pSTAT-6 levels in cocultures. Tumor cell–macrophage interaction induced STAT6 activation (N = 3).

Necrosis score in relation to other MRI features: Pearson r coefficient and p values are indicated for each variable. Correlation between necrosis score and ITSS.

Analysis by qPCR of the mRNA levels of the stemness markers CD133, EPHA2, NANOG, OCT 3 and 4, SNAIL, SOX2, ZEB1 in GB138 and GB83 spheroids (grey), compared to correspondent cells grown in adhesion (black).

Necrosis score and immunophenotyping values: Pearson r coefficient and p values are indicated for each variable. Linear correlation of necrosis score with tumor PDL-1 expression and PDL-1/FKBP51s PB-TAMs.

Immunoblot (IB) of pSTAT3 expression levels upon FKBP51s modulation in glioblastoma cell lines. (a) IB of U87 cell transfected with Flag-FKBP51, Flag-FKBP51s or correspondent EV as control. The spliced FKBP51s (short), but not the canonical FKBP51 (long), increased pSTAT3 levels. (b) IB of D54 cell transfected with siFKBP51s or a non-silencing RNA (NS) as control. FKBP51s silencing reduced pSTAT 3 levels (c) IB of GB83 cells transfected with siFKBP51s or a non-silencing RNA (NS) as control and with Flag-FKBP51s or correspondent EV as control. Densitometric analysis of the two blots are shown. Bands were quantitated by densitometry, using ImageJ 1.42q for Macintosh. Integrated optical densities (ODs) were normalized to a relative housekeeping gene and expressed as fold increase, arbitrarily using the control of each experiment as reference sample (expression =1).

IB of the expression levels of FKBP51s in 7 GB tumor cell lysates with the respective densitometric analysis of IB. Bands were quantitated by densitometry, using ImageJ 1.42q for Macintosh. Integrated ODs were normalized to two relative housekeeping genes (G3PDH and Tubulin).