Isolierte mikrovaskuläre Fragmente aus Fettgewebe als Vaskularisierungseinheiten im Tissue Engineering: Etablierung neuer Strategien zur Verbesserung ihrer Nutzbarkeit und ihres Vaskularisierungspotentials

Das Ziel des Tissue Engineerings ist die Herstellung von autologem Ersatzgewebe. Hierzu werden Zellen auf Trägermatrizes, die man als Scaffolds bezeichnet, gesiedelt. Die so entstandenen Gewebekonstrukte können anschließend in einen Gewebedefekt implantiert werden. Für den klinischen Erfolg dieser Methode ist die rasche Vaskularisierung der Implantate entscheidend. Dieser Prozess kann durch die Prävaskularisierung von Scaffolds mit adipösen mikrovaskulären Fragmenten, die sich über Inoskulation mit den Gefäßen des umliegenden Empfängergewebes verbinden, erheblich beschleunigt und verbessert werden. Die Ergebnisse vorangehender Studien weisen auf eine deutlich eingeschränkte Vaskularisierungskapazität von mikrovaskulären Fragmenten aus alten Spendern hin. Im ersten Studienabschnitt der vorliegenden Arbeit wurde daher zur möglichen Lösung dieses Problems erstmalig das Biobanking von mikrovaskulären Fragmenten junger Spender etabliert. Hierzu wurden mikrovaskuläre Fragmente aus den epididymalen Fettpolstern von C57BL/6 Spendermäusen isoliert, bei -196 °C für 7 Tage kryokonserviert und morphologisch sowie funktionell mit frisch isolierten Fragmenten verglichen. Zusätzlich wurden zur Analyse der in vivo Vaskularisierungskapazität kryokonservierte sowie frisch isolierte mikrovaskuläre Fragmente aus transgenen C57BL/6-TgN(ACTB-EGFP)1Osb/J Spendermäusen auf Integra®-Scaffolds gesiedelt und in die Rückenhautkammern von Green Fluorescent Protein (GFP)-negativen C57BL/6 Wildtyp-Empfängermäusen implantiert. Die Entwicklung neuer Gefäßnetzwerke wurde mittels intravitaler Fluoreszenzmikroskopie, Histologie und Immunhistochemie über 14 Tage untersucht. Rasterelektronenmikroskopische und durchflusszytometrische Analysen zeigten eine unveränderte Morphologie und zelluläre Zusammensetzung der kryokonservierten Fragmente, während ihre Anzahl und Viabilität im Vergleich zu frisch isolierten Kontrollen reduziert war. Die Kryokonservierung steigerte weiterhin die Expression pro-angiogener Proteine innerhalb der Fragmente, so dass sie auch mehr Wachstumsfaktoren sezernierten. Entsprechend verbanden sich sowohl die kryokonservierten als auch die frisch isolierten mikrovaskulären Fragmente innerhalb der implantierten Integra®-Scaffolds rasch zu durchbluteten Gefäßnetzwerken, welche sich in ihrer Dichte und mikrohämodynamischen Parametern nicht unterschieden. Dabei konnte die gesteigerte angiogene Aktivität der kryokonservierten Fragmente immunhistochemisch anhand eines größeren Anteils GFP-positiver Gefäße innerhalb der Netzwerke gezeigt werden. Es kann daher geschlussfolgert werden, dass die Kryokonservierung einen vielversprechenden Ansatz zur Langzeitlagerung mikrovaskulärer Fragmente darstellt. Die Ausbildung eines Gefäßnetzwerks durch mikrovaskuläre Fragmente benötigt trotz der ausgezeichneten regenerativen Eigenschaften dieser Vaskularisierungseinheiten mehrere Tage. Daher wurde im zweiten Teil der vorliegenden Arbeit untersucht, ob dieser Prozess durch Erythropoietin (EPO) weiter verbessert und beschleunigt werden kann. Hierzu wurden GFP-positive mikrovaskuläre Fragmente für 24 h in Medium kultiviert, welches mit EPO oder Vehikel supplementiert wurde. Anschließend erfolgte der Transfer der mikrovaskulären Fragmente auf Integra®-Scaffolds, welche in die Rückenhautkammern von GFP-negativen C57BL/6 Wildtyp-Empfängermäusen implantiert wurden. Mittels intravitaler Fluoreszenzmikroskopie sowie histologischen und immunhistochemischen Analysen wurde die Ausbildung neuer Gefäßnetzwerke über 14 Tage untersucht. EPO verbesserte die Viabilität und Proliferation der Fragmente und induzierte die Expression pro-angiogener Proteine. Entsprechend bildeten sich in den Scaffolds mikrovaskuläre Netzwerke deutlich schneller und dichter aus als in der Kontrollgruppe. Die beschleunigte Netzwerkbildung ging mit einer verbesserten Reifung der Gefäße und einer vermehrten Anzahl von GFP-positiven sowie -negativen Gefäßen im Randbereich um die Implantate einher. Somit kann aus den vorliegenden Ergebnissen geschlossen werden, dass EPO die Vaskularisierungskapazität mikrovaskulärer Fragmente steigert. Zusammenfassend zeigt die vorliegende Arbeit neue Ansätze auf, durch die der Einsatz von mikrovaskulären Fragmenten als Vaskularisierungseinheiten weiter verbessert werden kann. Diese Ansätze können zukünftig wesentlich zum klinischen Erfolg neuer Prävaskularisierungsstrategien im Tissue Engineering beitragen.Tissue engineering aims on the generation of autologous tissue substitutes. For this purpose, cells are seeded onto carrier matrices, also referred to as scaffolds. The resulting tissue constructs can be implanted into a tissue defect. The rapid vascularization of the implants is crucial for the clinical success of this approach. This can be achieved by the prevascularization of scaffolds with microvascular fragments. These microvascular fragments reassemble into new microvascular networks and develop interconnections to the host microvasculature. Recent studies indicate a reduced vascularization capacity of microvascular fragments from aged donors. To overcome this problem, biobanking of microvascular fragments from young donors has been established in the first part of the present thesis. Microvascular fragments were isolated from epididymal fat pads of C57BL/6 donor mice, cryopreserved at -196 °C for 7 days and subsequently compared with freshly isolated microvascular fragments. Moreover, cryopreserved and freshly isolated microvascular fragments from C57BL/6-TgN(ACTB-EGFP)1Osb/J donor mice were seeded on Integra® scaffolds, which were implanted into dorsal skinfold chambers of green fluorescent protein (GFP)-negative recipient animals. The formation of vascular networks within the implants was analyzed by means of intravital fluorescence microscopy, histology and immunohistochemistry throughout an observation period of 14 days. Scanning electron microscopy and flow cytometry revealed that cryopreservation did not affect the orphology and cellular composition of microvascular fragments, whereas their viability and number were significantly reduced when compared to freshly isolated controls. In addition, cryopreservation elevated the expression level of pro-angiogenic proteins within the microvascular fragments, which resulted in an increased release of growth factors. Accordingly, cryopreserved and control microvascular fragments induced a comparable vascularization of Integra® scaffolds without differences in microvascular network density, maturation, and remodeling. The enhanced angiogenic activity of cryopreserved microvascular fragments even resulted in a higher fraction of GFP-positive microvessels within the implants. These findings indicate that cryopreservation represents a promising concept for the long-term storage of microvascular fragments. Despite the excellent regenerative potential of microvascular fragments, their reassembly into blood-perfused vascular networks still requires several days. The second part of the present thesis analyzed whether this process may be further improved by erythropoietin (EPO). For this purpose, GFP-positive microvascular fragments were cultivated for 24 h in medium supplemented with EPO or vehicle. Subsequently, the microvascular fragments were seeded onto Integra® scaffolds, which were implanted into dorsal skinfold chambers of GFP-negative C57BL/6 recipient mice. The vascularization of the implants was studied by means of intravital fluorescence microscopy, histology and immunohistochemistry throughout an observation period of 14 days. EPO increased the viability and proliferation of microvascular fragments and upregulated their expression of pro-angiogenic proteins. Accordingly, the in vivo formation of vascular networks within implanted scaffolds seeded with EPO-treated microvascular fragments was improved, as indicated by a higher functional microvessel density when compared to controls. This enhanced vascularization was associated with an accelerated microvascular maturation. Moreover, an increased number of GFP-positive and -negative microvessels was detected within the tissue surrounding the scaffolds seeded with EPO-treated microvascular fragments. These findings indicate that EPO further boosts the vascularization capacity of microvascular fragments. Taken together, the present thesis demonstrates new approaches for an improved application of microvascular fragments as vascularization units. In the future, these approaches may markedly contribute to the clinical success of novel prevascularization strategies in tissue engineering.Deutsche Forschungsgemeinschaft - LA 2682/7‐

Prognostic validation of a new classification system for extent of resection in glioblastoma: a report of the RANO resect group

BACKGROUND Terminology to describe extent of resection in glioblastoma is inconsistent across clinical trials. A surgical classification system was previously proposed based upon residual contrast-enhancing (CE) tumor. We aimed to (I) explore the prognostic utility of the classification system and (II) define how much removed non-CE tumor translates into a survival benefit. METHODS The international RANO resect group retrospectively searched previously compiled databases from seven neuro-oncological centers in the USA and Europe for patients with newly diagnosed glioblastoma per WHO 2021 classification. Clinical and volumetric information from pre- and post-operative MRI were collected. RESULTS We collected 1008 patients with newly diagnosed IDHwt glioblastoma. 744 IDHwt glioblastomas were treated with radiochemotherapy per EORTC 26981/22981 (TMZ/RT→TMZ) following surgery. Among these homogenously treated patients, lower absolute residual tumor volumes (in cm 3) were favorably associated with outcome: patients with 'maximal CE resection' (class 2) had superior outcome compared to patients with 'submaximal CE resection' (class 3) or 'biopsy' (class 4). Extensive resection of non-CE tumor (≤5 cm 3 residual non-CE tumor) was associated with better survival among patients with complete CE resection, thus defining class 1 ('supramaximal CE resection'). The prognostic value of the resection classes was retained on multivariate analysis when adjusting for molecular and clinical markers. CONCLUSIONS The proposed "RANO categories for extent of resection in glioblastoma" are highly prognostic and may serve for stratification within clinical trials. Removal of non-CE tumor beyond the CE tumor borders may translate into additional survival benefit, providing a rationale to explicitly denominate such 'supramaximal CE resection'

Insulin-like growth factor 1 stimulates the angiogenic activity of adipose tissue-derived microvascular fragments

Angiogenesis in adipose tissue is promoted by insulin-like growth factor 1 signaling. We analyzed whether this regulatory mechanism also improves the angiogenic activity of adipose tissue-derived microvascular fragments. Murine adipose tissue-derived microvascular fragments were cultivated for 24 h in the University of Wisconsin (UW) solution supplemented with vehicle, insulin-like growth factor 1, or a combination of insulin-like growth factor 1 and insulin-like growth factor-binding protein 4. Subsequently, we assessed their cellular composition, viability, proliferation, and growth factor expression. Moreover, cultivated adipose tissue-derived microvascular fragments were seeded onto collagen-glycosaminoglycan scaffolds, which were implanted into dorsal skinfold chambers to study their vascularization and incorporation. Insulin-like growth factor 1 increased the viability and growth factor expression of adipose tissue-derived microvascular fragments without affecting their cellular composition and proliferation. Accordingly, scaffolds containing insulin-like growth factor 1-stimulated adipose tissue-derived microvascular fragments exhibited an enhanced in vivo vascularization and incorporation. These positive insulin-like growth factor 1 effects were reversed by additional exposure of adipose tissue-derived microvascular fragments to insulin-like growth factor-binding protein 4. Our findings indicate that insulin-like growth factor 1 stimulation of adipose tissue-derived microvascular fragments is suitable to improve their vascularization capacity

PCV chemotherapy alone for WHO grade 2 oligodendroglioma: prolonged disease control with low risk of malignant progression

INTRODUCTION The role of chemotherapy alone in newly diagnosed WHO grade 2 oligodendroglioma after biopsy, incomplete or gross total resection remains controversial. We here analyze the clinical outcome of four patient cohorts being treated with either procarbazine, CCNU and vincristine (PCV) or temozolomide (TMZ) after biopsy, resection only, or wait-and-scan after biopsy. METHODS Patients (n = 142) with molecularly defined oligodendroglioma (WHO 2016) were assigned to four cohorts: W&S, wait-and-scan after stereotactic biopsy (n = 59); RES, surgical resection only (n = 27); TMZ, temozolomide after biopsy (n = 26) or PCV (n = 30) after biopsy. Presurgical MRI T2 tumor volumes were obtained by manual segmentation. Progression-free survival (PFS), post-recurrence PFS (PR-PFS) and rate of histological progression to grade 3 were analyzed. RESULTS PFS was longest after PCV (9.1 years), compared to 5.1 years after W&S, 4.4 years after RES and 3.6 years after TMZ. The rate of histological progression from grade 2 to 3 within 10 years was 9% for the PCV, 29% for the W&S, 67% for the RES and 75% for the TMZ group (p = 0.01). In the W&S group, patients treated with PCV at first relapse had a longer PFS from intervention than those treated with TMZ (7.2 vs 4.0 years, p = 0.04). Multivariate analysis identified smaller tumor volume prior to any intervention (p = 0.02) to be prognostic for PFS. CONCLUSIONS PCV chemotherapy alone is an effective treatment for WHO grade 2 oligodendroglioma, with long PFS and low rate of histological progression

Past medical history of tumors other than meningioma is a negative prognostic factor for tumor recurrence in meningiomas WHO grade I

BACKGROUND Prognostic markers for meningioma recurrence are needed to guide patient management. Apart from rare hereditary syndromes, the impact of a previous unrelated tumor disease on meningioma recurrence has not been described before. METHODS We retrospectively searched our database for patients with meningioma WHO grade I and complete resection provided between 2002 and 2016. Demographical, clinical, pathological, and outcome data were recorded. The following covariates were included in the statistical model: age, sex, clinical history of unrelated tumor disease, and localization (skull base vs. convexity). Particular interest was paid to the patients' past medical history. The study endpoint was date of tumor recurrence on imaging. Prognostic factors were obtained from multivariate proportional hazards models. RESULTS Out of 976 meningioma patients diagnosed with a meningioma WHO grade I, 416 patients fulfilled our inclusion criteria. We encountered 305 women and 111 men with a median age of 57 years (range: 21-89 years). Forty-six patients suffered from a tumor other than meningioma, and no TERT mutation was detected in these patients. There were no differences between patients with and without a positive oncological history in terms of age, tumor localization, or mitotic cell count. Clinical history of prior tumors other than meningioma showed the strongest association with meningioma recurrence (p = 0.004, HR = 3.113, CI = 1.431-6.771) both on uni- and multivariate analysis. CONCLUSION Past medical history of tumors other than meningioma might be associated with an increased risk of meningioma recurrence. A detailed pre-surgical history might help to identify patients at risk for early recurrence

Extent, pattern, and prognostic value of MGMT promotor methylation: does it differ between glioblastoma and IDH-wildtype/TERT-mutated astrocytoma?

INTRODUCTION The cIMPACT-NOW update 6 first introduced glioblastoma diagnosis based on the combination of IDH-wildtype (IDHwt) status and TERT promotor mutation (pTERTmut). In glioblastoma as defined by histopathology according to the WHO 2016 classification, MGMT promotor status is associated with outcome. Whether this is also true in glioblastoma defined by molecular markers is yet unclear. METHODS We searched the institutional database for patients with: (1) glioblastoma defined by histopathology; and (2) IDHwt astrocytoma with pTERTmut. MGMT promotor methylation was analysed using methylation-specific PCR and Sanger sequencing of CpG sites within the MGMT promotor region. RESULTS We identified 224 patients with glioblastoma diagnosed based on histopathology, and 54 patients with IDHwt astrocytoma with pTERTmut (19 astrocytomas WHO grade II and 38 astrocytomas WHO grade III). There was no difference in the number of MGMT methylated tumors between the two cohorts as determined per PCR, and also neither the number nor the pattern of methylated CpG sites differed as determined per Sanger sequencing. Progression-free (PFS) and overall survival (OS) was similar between the two cohorts when treated with radio- or chemotherapy. In both cohorts, higher numbers of methylated CpG sites were associated with favourable outcome. CONCLUSIONS Extent and pattern of methylated CpG sites are similar in glioblastoma and IDHwt astrocytoma with pTERTmut. In both tumor entities, higher numbers of methylated CpG sites appear associated with more favourable outcome. Evaluation in larger prospective cohorts is warranted

Extent and prognostic value of MGMT promotor methylation in glioma WHO grade II

MGMT promotor methylation is associated with favourable outcome in high-grade glioma. In glioma WHO grade II, it is unclear whether the extent of MGMT promotor methylation and its prognostic role is independent from other molecular markers. We performed a retrospective analysis of 155 patients with glioma WHO grade II. First, all 155 patients were assigned to three molecular groups according to the 2016 WHO classification system: (1) oligodendroglioma, IDH-mutant and 1p19q co-deleted (n=81);(2) astrocytoma, IDH-mutant and 1p19q non-codeleted (n=54);(3) astrocytoma, IDH-wildtype (n=20). MGMT promotor methylation was quantified using Sanger sequencing of the CpG sites 74-98 within the MGMT promotor region. Highest numbers of methylated CpG sites were found for oligodendroglioma, IDH-mutant and 1p19q co-deleted. When 1p19q co-deletion was absent, numbers of methylated CpG sites were higher in the presence of IDH-mutation. Accordingly, lowest numbers were seen in the IDH-wildtype subpopulation. In the entire cohort, larger numbers of methylated CpG sites were associated with favourable outcome. When analysed separately for the three WHO subgroups, a similar association was only retained in astrocytoma, IDH-wildtype. Collectively, extent of MGMT promotor methylation was strongly associated with other molecular markers and added prognostic information in astrocytoma, IDH-wildtype. Evaluation in prospective cohorts is warranted

Defining Treatment‐Related Adverse Effects in Patients with Glioma: Distinctive Features of Pseudoprogression and Treatment‐Induced Necrosis

Background: Pseudoprogression (PP) and treatment‐induced brain tissue necrosis (TN) are challenging cancer treatment–related effects. Both phenomena remain insufficiently defined; differentiation from recurrent disease frequently necessitates tissue biopsy. We here characterize distinctive features of PP and TN to facilitate noninvasive diagnosis and clinical management. Materials and Methods: Patients with glioma and confirmed PP (defined as appearance 5 months after RT) were retrospectively compared using clinical, radiographic, and histopathological data. Each imaging event/lesion (region of interest [ROI]) diagnosed as PP or TN was longitudinally evaluated by serial imaging. Results: We identified 64 cases of mostly (80%) biopsy‐confirmed PP (n = 27) and TN (n = 37), comprising 137 ROIs in total. Median time of onset for PP and TN was 1 and 11 months after RT, respectively. Clinically, PP occurred more frequently during active antineoplastic treatment, necessitated more steroid‐based interventions, and was associated with glioblastoma (81 vs. 40%), fewer IDH1 mutations, and shorter median overall survival. Radiographically, TN lesions often initially manifested periventricularly (n = 22/37; 60%), were more numerous (median, 2 vs. 1 ROIs), and contained fewer malignant elements upon biopsy. By contrast, PP predominantly developed around the tumor resection cavity as a non‐nodular, ring‐like enhancing structure. Both PP and TN lesions almost exclusively developed in the main prior radiation field. Presence of either condition appeared to be associated with above‐average overall survival. Conclusion: PP and TN occur in clinically distinct patient populations and exhibit differences in spatial radiographic pattern. Increased familiarity with both conditions and their unique features will improve patient management and may avoid unnecessary surgical procedures. Implications for Practice: Pseudoprogression (PP) and treatment‐induced brain tissue necrosis (TN) are challenging treatment‐related effects mimicking tumor progression in patients with brain cancer. Affected patients frequently require surgery to guide management. PP and TN remain arbitrarily defined and insufficiently characterized. Lack of clear diagnostic criteria compromises treatment and may adversely affect outcome interpretation in clinical trials. The present findings in a cohort of patients with glioma with PP/TN suggest that both phenomena exhibit unique clinical and imaging characteristics, manifest in different patient populations, and should be classified as distinct clinical conditions. Increased familiarity with PP and TN key features may guide clinicians toward timely noninvasive diagnosis, circumvent potentially unnecessary surgical procedures, and improve response assessment in neuro‐oncology