TGFß-blockade uncovers stromal plasticity in tumors by revealing the existence of a subset of interferon-licensed fibroblasts.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFß in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFß and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

Phenotypic characterization of isolated valvular interstitial cell subpopulations.

BACKGROUND AND AIM OF THE STUDY: Valvular interstitial cells (VICs) demonstrate a heterogeneous range of phenotypes such as variable expression of smooth muscle alpha-actin (SMalphaA). Myofibroblast-like VICs, expressing high levels of SMalphaA, are thought to be involved in myxomatous degeneration of mitral valves. The inability to isolate specific cell types has restricted potential investigations of valvular disease mechanisms. Thus, investigations were conducted into methods of isolating different cell subpopulations from primary VICs as a preparatory step for cell type-specific evaluations of heart valve disease. METHODS: VICs were isolated from porcine valves, cultured to 80% confluency, and subdivided using differential detachment or adhesion. The subdivided cells were further cultured and analyzed phenotypically by immunocytochemistry and flow cytometry to characterize SMalphaA expression. Roundness and growth rates were also analyzed. RESULTS: VICs that were relatively sensitive to trypsinization expressed low and heterogeneous levels of SMalphaA (15-35%), whereas more-adherent VICs expressed higher and homogeneous levels (>98%) suggestive of a myofibroblast-like phenotype. The more-adherent cells also had lower growth potential and were less round than less-adhesive VICs. Separated cell subtypes were found to maintain their phenotype through several cell passages. CONCLUSION: VICs are a mixed population of cells, many of which express high levels of SMalphaA. Differential detachment and adhesion can effectively separate cell subpopulations from primary cultures of VICs. The ability to study valve cell subpopulations has substantial implications for future analyses of valvular biology, disease, and tissue engineering.

Studying clonal dynamics in response to cancer therapy using high-complexity barcoding.

Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.

Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations.

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of all meningiomas, but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas had simple genomes, with fewer mutations, rearrangements and copy-number alterations than reported in other tumors in adults. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements, including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers in an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (p.Glu17Lys) and SMO (p.Trp535Leu) and exhibited immunohistochemical evidence of activation of these pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets.

Polymorphisms in TGFbeta and TNFalpha are associated with the myelodysplastic syndrome phenotype.

CONTEXT: Myelodysplastic syndromes (MDSs) are characterized by ineffective hematopoiesis, excessive apoptosis, and the aberrant expression of a number of cytokines. The genes encoding these cytokines are significantly polymorphic. It is unknown whether these cytokine polymorphisms are associated with, and may therefore be playing a role in the pathogenesis of, MDS. OBJECTIVE: To determine if certain polymorphisms in the tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) cytokines are overrepresented in a cohort of patients with MDSs. DESIGN: DNA was isolated from the peripheral blood or bone marrow aspirate of 21 patients with MDS. The genotypes for 4 different polymorphisms, 2 in TNFalpha and 2 in TGFbeta1, were determined using single-specific-primer polymerase chain reaction. The allele and genotype frequencies were compared with similar populations in the National Cancer Institute SNP500 database. RESULTS: In our MDS population, the -308A/A genotype of the TNFalpha gene and the TGFbeta1 allele +29T and genotype +29T/T, each associated with higher levels of expression, were overrepresented in our MDS population. CONCLUSIONS: Polymorphisms associated with increased expression in the cytokines TNFalpha and TGFbeta1 are overrepresented in the MDS population suggesting that increased TNF-alpha and TGF-beta1 activity may contribute to the susceptibility and/or pathogenesis of MDS. Further studies with larger sample sizes are warranted to confirm our observation.