Ultrastructural Characterization of Primary Cilia in Pathologically Characterized Human Glioblastoma Multiforme (GBM) Tumors

Joanna J Moser; Marvin J Fritzler; Jerome B Rattner

Disclosures

BMC Clin Pathol. 2014;14(40) 

In This Article

Results

We examined both formalin-fixed paraffin embedded (FFPE) and fresh-fixed tissue from seven cases of surgically resected brain tumors diagnosed by neuropathologists as grade IV glioblastoma/GBM using indirect immunofluorescence (IIF) and electron microscopy (EM), respectively.

The GBM tumors were molecularly characterized according to IDH 1/2 mutation status, EGFR amplification status and MGMT promoter status (Table 1). Our results showed that 71% of GBM patients had amplified levels of EGFR, 86% had no IDH1/2 mutations and 50% had methylated MGMT promoters (Table 1).

We examined the biopsy tissue from each of the 7 patients by light and electron microscopy. IIF examination of tissue from patient #1 showed typical primary cilia (Figure 2, top inset). Similarly, ultrastructural examination revealed a normal basal body with a fully formed mature primary cilium, consistent with normal morphology (Figure 2 compared to Figure 1). The cilium-pit was well defined (Figure 2) and the cilium contained well-formed microtubules with normal spacing between doublets (Figure 2, bottom inset). In addition, small vesicles were seen along the cilium microtubules and interfacing with the cilium-pit (Figure 2), which is consistent with previous findings that showed this is a site for endocytosis based signalling.[17]

Figure 2.

Patient #1. GBM cells have an intact primary cilium. Electron micrograph showing a mature primary cilium and basal body with a well-formed cilium-pit (arrow) and endocytotic vesicles (short arrows). Top inset, primary cilia as marked by acetylated tubulin (green) using indirect immunofluorescence analysis. Bottom inset, cross section through the axoneme of another cell. EM scale bar = 100 nm, IIF scale bar = 7 μm.

The GBM tissue from patient #2 failed to show abundant primary cilia by IIF. Ultrastructural examination revealed cells with basal bodies reminiscent of stage 1 ciliogenesis, however there were no paired vesicles present along the lateral sides of the distal end of the basal body/transition zone as observed in longitudinal and cross-sections (Figure 3A and inset compared to Figure 1). In addition to missing paired vesicles, patient #2 had basal bodies that presented with abnormal, vertically outstretched distal appendages (Figure 3B). Figure 3C shows another example of an abnormal basal body with absent paired vesicles along the lateral sides of the distal end of the basal body/transition zone. Interestingly, the transition zone plate is present without the presence of the vesicles which suggests that the vesicles do not need to be present to allow the transition zone to form, but need to be present to allow ciliogenesis to progress beyond stage 1. In one rare example, a primary cilium which had progressed to stage 5 of ciliogenesis was found (Figure 3D). On close examination, this cilium displayed a disrupted ciliary membrane which was also the site of cytoplasmic extrusions into the surrounding environment (Figure 3D). These abnormal primary cilia also have a dark pericentriolar material (PCM)-like collection of material clumped along the transition zone of the primary cilium with darkening of the cilia microtubules at the proximal end of the cilium shaft (Figure 3D).

Figure 3.

Patient #2. GBM cells are halted at stage 1 of ciliogenesis with rare cells progressing to stage 5. (A) Basal body and abnormal stage 1 cilium with absent paired lateral vesicles. Inset cross section through the transition zone from another cell. (B) Basal body and abnormal stage 1 cilium with vertically outstretched distal appendages and no vesicles. (C) Basal body with clear transition zone and abnormal stage 1 cilium with absent vesicles. (D) Rare occurrence of a primary cilium at stage 5 with abnormal destruction of the cilium-pit with cytoplasmic extrusion, darkened microtubules at proximal end of cilium and electron dense collection of material at the transition zone. EM scale bars = 500 nm.

The tissue from patient #3 revealed an absence of mature primary cilia by IIF and ultrastructural examination showed that 70% of centrosome/basal body profiles were at stage 1 while the remaining 30% of profiles examined displayed stage 2/3 of ciliogenesis (Figure 4). Many of the immature cilia contained electron dense material along the cilium shaft (Figure 4 compared to Figure 1). The stretched vesicular hat that is so prominent in normal cells at stage 2/3 was irregular, thin and misshapen in patient #3 GBM cells (Figure 4). The microtubules of the cilium appear irregular, lack organization and do not display the normal architectural characteristics of the transition zone (Figure 4). There were no cilia in stages 4–5 observed for patient #3.

Figure 4.

Patient #3. GBM cells are halted at stage 3 of ciliogenesis and display electron dense material clustered along the cilium shaft (arrow heads) and an irregular vesicular hat (arrows). EM scale bar = 100 nm.

GBM tissue from patient #4 failed to display mature primary cilium by IIF. Ultrastructurally, patient #4 expressed basal bodies that were similar to stage 1 with a transition zone plate formed along the distal end (Figure 5A compared to Figure 1). It is important to note that in many of these electron microscope centrosome/basal body profiles there was only 1 vesicle present (as opposed to the normal 2 vesicles) at the distal end and the vesicle was positioned directly above the transition zone plate (as opposed to the normal lateral orientation beside the transition zone plate) (Figure 5A). The architecture of the distal appendages along the basal body was also abnormal given their outstretched vertical appearance (Figure 5A) as opposed to the normal horizontal appearance displayed in Figure 1. Figure 5B, illustrates a discontinuity present in one of the centriole microtubule blades, although this centriole was 357 nm in length (Figure 5B) which falls within normal parameters.[17]

Figure 5.

Patient #4. GBM cells were characterized by (A) primary cilia that were halted at stage 1 of ciliogenesis with a single vesicle present above the transition plate and abnormal distal appendages and (B) breakages in the basal body/centriole microtubules. EM scale bars = 100 nm.

Samples from patient #5 did not show mature primary cilia by IIF. Ultrastructural examination revealed either basal bodies with an immature transition zone or profiles similar to stage 1 (Figure 6 compared to Figure 1). The transition zone was not visible in the electron micrograph centrosome/basal body profiles from this patient and we did not observe any paired vesicles similar to that seen in patient #2 (Figure 6). There also appears to be minimal PCM distributed in the cytoplasmic area surrounding the basal body and centriole (Figure 6).

Figure 6.

Patient #5. GBM cells were characterized by abnormal primary cilia that were halted at or before stage 1 of ciliogenesis with no evidence of paired vesicles or a transition zone plate. EM scale bar = 250 nm.

There were no observable primary cilia staining by IIF in the GBM tissue from patient #6, although ultrastructural examination showed cilia at multiple stages (Figure 7). We observed profiles at stage 1 with a well-defined plate within the transition zone lacking paired laterally placed vesicles (Figure 7A). We observed many cilia at stage 1 with either multiple irregular abnormally shaped vesicles formed at the distal end of the basal body or 4 distinct vesicles above the plate within the transition zone (Figure 7B). In the latter case, distal appendages were absent. In rare cases, a cilium with a short axoneme was detected (Figure 7C). These cilia appeared to have a truncated cilium-pit so that the distal end of the cilium is continuous with the cytoplasm, a configuration reminiscent of a regressing cilium.[26]

Figure 7.

Patient #6. GBM cells were characterized by abnormal primary cilia at (A, B) stage 1/2 and (C) 4/5 of ciliogenesis with either absent lateral vesicles, aberrant supernumerary vesicles along the length of the transition zone plate or abrupt cessation of the cilium shaft. EM scale bars = 100 nm.

GBM tissue from patient #7 also did not show any primary cilia by IIF staining. Ultrastructurally, primary ciliogenesis occurred in profiles to a maximum of stage 2 (Figure 8 compared to Figure 1). These cells had vesicular hats that were misshaped and swollen (Figure 8A and B) and had outstretched, vertical distal basal body appendages (Figure 8B). These characteristics were similar to those observed in other astrocytoma/glioblastoma cell lines, particularly in U-373 MG and U-138 MG cells (Figure 2B in[17]).

Figure 8.

Patient #7. GBM cells were characterized by abnormal primary cilia at stage 2 of ciliogenesis with (A) swollen vesicular hats (B) misshaped vesicular hats and abnormal distal basal body appendages. EM scale bars = 100 nm.

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