Patients and clinical data
Patient-derived cell lines at Turku University Hospital and the glioma tissue microarrays (TMAs) with patient follow-up data were established with permission from the Auria Biobank steering committee and the Hospital District of Southwest Finland. For cell culture, all patients had given a written Biobank consent statement. The samples for TMAs and associated data were obtained as described previously [10], in accordance with the Finnish Biobank Act (688/2012) which does not require a separate informed consent from individual patients.
Transcriptomic analysis
As a pilot study, we utilized data from a transcriptomic analysis of migrating primary glioblastoma cells compared to cells grown as spheroids from a previous study [2]. To summarize the experiment the data originated from, five cell lines (T111, T113, T78, T86 and T87) were established from glioblastoma tumor samples and grown in serum free conditions as spheroids. Spheroids had been plated in a semisolid matrix where part of the cells differentiated into elongated migrating cells. The migrating cells had been mechanically separated from non-migrating cells. RNA from migrating cells and free floating spheroids had been subjected to array-based transcriptomic analysis (Affymetrix 133 Plus 2.0 microarray). From the normalized data, we extracted the expression data of all 15 formin genes Formin 1 (FMN1), Formin 2 (FMN2), Disheveled-associated activator of morphogenesis 1 (DAAM1), Disheveled-associated activator of morphogenesis 2 (DAAM2), diaphanous related formin 1 (mDia1), diaphanous related formin 3 (mDia2), diaphanous related formin 2 (mDia3), FHOD1, Formin Homology 2 Domain Containing 3 (FHOD3), Formin-like protein 1 (FMNL1), Formin-like protein 2 (FMNL2); Formin-like protein 3 (FMNL3), Delphilin or delta 2-interacting protein 1 (GRID2IP), Inverted formin 1 (INF1), and INF2 and investigated their expression as fold-change in migrating cells compared to spheroid cells. Over 2 fold-change was considered upregulation in migrating cells.
Cell culture
The commercially available glioblastoma human cell lines U87, U138 and T98 were from American Type Culture Collection (Manassas, VA, USA; catalogue numbers HTB-14, HTB-16 and CRL-1690, respectively). U138 and T98 were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 5 mM ultraglutamine and 100 U/ml penicillin-streptomycin (Gibco, CA, USA), while U87 was kept in spheroid medium (DMEM/F12, 1% B27, 100 U/ml penicillin-streptomycin (Gibco), 40 ng/ml fibroblast growth factor-2 (FGF-2; Gibco), and 40 ng/ml epidermal growth factor (EGF; Invitrogen, CA, USA).
Primary glioblastoma cell cultures established from glioblastoma samples at Turku University Hospital were named University of Turku Glioblastoma (UTGB). Following surgical resection, approximately 1–2 g of tumor tissue was collected and cultured as described by Rustenhoven et al. [11] From 10 tumor samples, 3 cell lines were established and tested negative for mycoplasma. The cell lines had the same mutations as the original tumor as tested by a 20 gene NGS panel designed for glioma diagnostics [12]. One cell line did not form spheroids, and was omitted from further studies. The clinicopathological characteristics of the two remaining primary cell lines UTGB4 and UTGB7 are described in Supplemental Table 1. In addition to these primary cells lines, we obtained T78, T86, and T87 primary glioblastoma cell lines that were included in the transcriptomics analysis [2]. All cell lines were repeatedly tested for mycoplasma contamination, and remained negative.
Spheroid formation
Spontaneous spheroid formation was observed for U87, T86 and UTGB7 cells 3 to 6 days after plating single cells in spheroid medium. For all the other cell lines, spheroid formation was achieved using the hanging-drop technique for 48 h in 6 μl spheroid medium (UTGB4, 4 × 103 cells/drop; T78 and T87, 2 × 103 cells/drop) or DMEM 10% FBS (T98 and U138, 2 × 103 cells/drop). Only spheroids with the diameter of 200–600 μm were used in the experiments.
Cell migration
In order to study if and which formins are up or downregulated in migrating cells, spheroids were allowed to adhere in Geltrex (hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix, Gibco) precoated 6-well plate (30–40 spheroids/well) during 24 h. Using a microscope, attached spheroids were separated with the help of a needle and recovered from the supernatant. Migrated cells were detached with trypsin-EDTA solution (Sigma-Aldrich), and pelleted by centrifugation.
qRT-PCR
Total RNA from different fractions was extracted using NucleoSpin RNA/Protein Kit (Macherey-Nagel, PA, USA) according to the manufacturer’s protocol and processed to cDNA with SensiFast cDNA Synthesis Kit (Bioline, OH, USA). TaqMan qRT-PCR was performed with a QuantStudio 12 K Flex Real-Time PCR System (Turku Centre for Biotechnology) using specific primers (Supplement 1) and quantitation was carried out with QuantStudio 12 K Flex software using the ΔΔCT method in order to calculate the relative fold gene expression. Three replicate samples were studied for detection of target mRNA expression and GAPDH was used as an endogenous control. The quantities were expressed as a fold change relative in migrating cells compared to immotile spheroids. The results are presented as means.
Transfection with small interfering RNAs, spheroid migration assay and stainings
The mDia1, mDia2, FHOD1,and INF2 transcripts were knocked down individually in the commercial cell lines U87 and U138, and primary glioblastoma cell lines T86 and UTGB7 using 50 nM of SMARTpool small interfering RNA (siRNA) (Dharmacon Research, Boulder, CA). Non-targeting Pool siRNA was used as control. Cells attached as monolayer on Geltrex precoated 12-well plates were transfected using Dharmafect 4 transfection reagent (Dharmacon Research) according to the manufacturer’s instructions. The knockdown efficacy was examined 48 h after transfection by western blotting as described elsewhere [13]. Antibodies, dilutions and spheroid staining procedures are described in Supplement 1.
Forty-eight hours after siRNA treatment, cells were trypsinized and plated as drops for spheroid formation for another 48 h. To analyze migration spheroids were plated in Geltrex precoated 96-well Image Lock plate (Essen Bioscience, Ann Arbor, MI) and imaged every 2 h using the Incucyte S3 incubator system (Essen Bioscience). Areas of migrated cells and spheroids were used to indicate migration fold increase related to time point 1. The experiment was repeated at least 3 times, with a total of 25–50 spheroids per cell line for each siRNA treatment. Differences in migration from spheroids were analyzed using Student t-test. Two-tailed p values ≤0.05 were regarded as significant.
Immunohistochemistry of glioblastoma samples
3.5 um sections from TMA:s composed of 1.5 mm cores of glioma samples were obtained from the Auria Biobank. The cohort includes diffuse glioma samples and has been previously described in detail [10]. It also includes relevant clinicopathological parameters and follow-up data. From this cohort, we analyzed the 93 samples that had an integrated diagnosis of glioblastoma, IDH-wildtype according to the WHO 2016 classification [14]. The slides were stained by the streptavidin-peroxidase method; using a Labvision staining device (Thermo Fisher Scientific, Fremont, CA) with a Bright Vision Poly-HRP-anti-mouse/rabbit kit (Immunologic, Duiven, the Netherlands) as described previously [15]. Briefly, antigen retrieval for INF2 staining was carried out by microwaving the slides in a pH 9 buffer. Primary antibody dilutions used were 1:75 for anti-FHOD1 (Sigma-Aldrich, St Louis, MA; catalogue number HPA024468) and 1:500 for INF2 (Proteintech, Chicago, IL; catalogue number 20466–1-AP). Furthermore, 10 whole slide samples were evaluated to compare FHOD1 and INF2 expression in tumor bulk to areas of diffuse tumor cell infiltration in brain tissue. For this, 10 cases with immunohistochemically detected p53 positivity indicating TP53 mutation and representative areas of solid tumor and diffuse infiltration were selected. The p53 stainings were performed using Ventana reagents and a Ventana Benchmark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ).
Scoring was performed by two pathologists blinded to clinical data (MG and OC), with 0 standing for negative, 1 for weak, 2 for moderate and 3 for strong staining. Representative images of staining categories are presented in Fig. 3.
For survival analyses, both FHOD1 and INF2 staining scores were dichotomized into low expression (score 0 or 1) or high expression (score 2 or 3). Kaplan-Meier with log-rank test and univariate and multivariate Cox proportional hazards model were performed to assess survival. Multivariate Cox regression was analyzed using adjustments for age, pre-operative Karnofsky Performance Scale (KPS), resection type, and post-operative adjuvant treatment. Overall survival (OS) was defined as the time from surgical resection to death or end of follow-up. Progression-free survival (PFS) was a composite end-point defined as the time from surgical resection to the first tumor progression indicated by re-resection, start of a new treatment regimen, death, or end of follow-up. Survival analyses were conducted using IBM SPSS Statistics version 25.0 for Mac (IBM Corp., Armonk, NY).