Blocking TGF-ß- and Epithelial-to-Mesenchymal Transition (EMT)-mediated activation of vessel-associated mural cells in glioblastoma impacts tumor angiogenesis.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. GBM displays excessive and unfunctional vascularization which may, among others, be a reason for its devastating prognosis. Pericytes have been identified as the major component of the irregular vessel structure in GBM. In vitro data suggest an epithelial-to-mesenchymal transition (EMT)-like activation of glioma-associated pericytes, stimulated by GBM-secreted TGF-ß, to be involved in the formation of a chaotic and dysfunctional tumor vasculature. This study investigated whether TGF-ß impacts the function of vessel associated mural cells (VAMCs) in vivo via the induction of the EMT transcription factor SLUG and whether this is associated with the development of GBM-associated vascular abnormalities. Upon preventing the TGF-ß-/SLUG-mediated EMT induction in VAMCs, the number of PDGFRß and αSMA positive cells was significantly reduced, regardless of whether TGF-ß secretion by GBM cells was blocked or whether SLUG was specifically knocked out in VAMCs. The reduced amount of PDGFRß+ or αSMA+ cells observed under those conditions correlated with a lower vessel density and fewer vascular abnormalities. Our data provide evidence that the SLUG-mediated modulation of VAMC activity is induced by GBM-secreted TGF-߬ and that activated VAMCs are key contributors in neo-angiogenic processes. We suggest that a pathologically altered activation of GA-Peris in the tumor microenvironment is responsible for the unstructured tumor vasculature. There is emerging evidence that vessel normalization alleviates tumor hypoxia, reduces tumor-associated edema and improves drug delivery. Therefore, avoiding the generation of an unstructured and non-functional tumor vasculature during tumor recurrence might be a promising treatment approach for GBM and identifies pericytes as a potential novel therapeutic target.

Functional Investigation of IGF1R Mutations in Multiple Myeloma.

High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) and RTK mutations are associated with high-risk/worse prognosis in multiple myeloma (MM). Combining the pIGF1R/pINSR inhibitor linsitinib with the proteasome inhibitor (PI) bortezomib seemed promising in a clinical trial, but IGF1R expression was not associated with therapy response. Because the oncogenic impact of IGF1R mutations is so far unknown, we investigated the functional impact of IGF1R mutations on survival signaling, viability/proliferation and survival response to therapy. We transfected four human myeloma cell lines (HMCLs) with IGF1RWT, IGF1RD1146N and IGF1RN1129S (Sleeping Beauty), generated CRISPR-Cas9 IGF1R knockouts in the HMCLs U-266 (IGF1RWT) and L-363 (IGF1RD1146N) and tested the anti-MM activity of linsitinib alone and in combination with the second-generation PI carfilzomib in seven HMCLs. IGF1R knockout entailed reduced proliferation. Upon IGF1R overexpression, survival signaling was moderately increased in all HCMLs and slightly affected by IGF1RN1129S in one HMCL, whereby the viability remained unaffected. Expression of IGF1RD1146N reduced pIGF1R-Y1135, especially under serum reduction, but did not impact downstream signaling. Linsitinib and carfilzomib showed enhanced anti-myeloma activity in six out of seven HMCL irrespective of the IGF1R mutation status. In conclusion, IGF1R mutations can impact IGF1R activation and/or downstream signaling, and a combination of linsitinib with carfilzomib might be a suitable therapeutic approach for MM patients potentially responsive to IGF1R blockade.

Modeling Myeloma Dissemination In Vitro with hMSC-interacting Subpopulations of INA-6 Cells and Their Aggregation/Detachment Dynamics.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSC) stimulate myeloma cell expansion (e.g., IL6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs cocultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNA sequencing, cell viability, and apoptosis. Results were correlated with gene expression data (n = 837) and survival of patients with myeloma (n = 536). On dispersed hMSCs, INA-6 saturate hMSC surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance. SIGNIFICANCE: Novel methods describe in vitro dissemination of myeloma cells as detachment of daughter cells after cell division. Myeloma adhesion genes were identified that counteract in vitro detachment with potential clinical relevance.