Predictive oncology: a review of multidisciplinary, multiscale in silico modeling linking phenotype, morphology and growth.

Empirical evidence and theoretical studies suggest that the phenotype, i.e., cellular- and molecular-scale dynamics, including proliferation rate and adhesiveness due to microenvironmental factors and gene expression that govern tumor growth and invasiveness, also determine gross tumor-scale morphology. It has been difficult to quantify the relative effect of these links on disease progression and prognosis using conventional clinical and experimental methods and observables. As a result, successful individualized treatment of highly malignant and invasive cancers, such as glioblastoma, via surgical resection and chemotherapy cannot be offered and outcomes are generally poor. What is needed is a deterministic, quantifiable method to enable understanding of the connections between phenotype and tumor morphology. Here, we critically assess advantages and disadvantages of recent computational modeling efforts (e.g., continuum, discrete, and cellular automata models) that have pursued this understanding. Based on this assessment, we review a multiscale, i.e., from the molecular to the gross tumor scale, mathematical and computational "first-principle" approach based on mass conservation and other physical laws, such as employed in reaction-diffusion systems. Model variables describe known characteristics of tumor behavior, and parameters and functional relationships across scales are informed from in vitro, in vivo and ex vivo biology. We review the feasibility of this methodology that, once coupled to tumor imaging and tumor biopsy or cell culture data, should enable prediction of tumor growth and therapy outcome through quantification of the relation between the underlying dynamics and morphological characteristics. In particular, morphologic stability analysis of this mathematical model reveals that tumor cell patterning at the tumor-host interface is regulated by cell proliferation, adhesion and other phenotypic characteristics: histopathology information of tumor boundary can be inputted to the mathematical model and used as a phenotype-diagnostic tool to predict collective and individual tumor cell invasion of surrounding tissue. This approach further provides a means to deterministically test effects of novel and hypothetical therapy strategies on tumor behavior.

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

EFFECTS OF THE ACTA2 R258C MUTATION ON VASCULAR SMOOTH MUSCLE CELL PHENOTYPE AND PROPERTIES

Thoracic Aortic Aneurysms and Dissections (TAAD) are the fifteenth leading cause of death in the United States. About 15% of TAAD patients have family history of the disease. The most commonly mutated gene in these families is ACTA2, encoding smooth muscle-specific α-actin. ACTA2 missense mutations predispose individuals both to TAAD and to vascular occlusive disease of small, muscular arteries. Mice carrying an Acta2 R258C mutant transgene with a wildtype Acta2 promoter were generated and bred with Acta2-/- mice to decrease the wildtype: mutant Acta2 ratio. Acta2+/+ R258C TGmice have decreased aortic contractility without aortic disease. Acta2+/- R258C TG mice, however, have significant aortic dilatations by 12 weeks of age and a hyperproliferative response to injury. We characterized smooth muscle cells (SMCs) from bothmouse models under the hypothesis that mutant α-actin has a dominant negative effect, leading to impaired contractile filament formation/stability, improper focal adhesion maturation and increased proliferation. Explanted aortic SMCs from Acta2+/+ R258C TG mice are differentiated - they form intact filaments, express higher levels of contractile markers compared to wildtype SMCs and have predominantly nuclear Myocardin-Related Transcription Factor A (MRTF-A) localization. However, ultracentrifugation assays showed large unpolymerized actin fractions, suggesting that the filaments are brittle. In contrast, Acta2+/- R258C TG SMCs are less well-differentiated, with pools of unpolymerized actin, more cytoplasmic MRTF-A and decreased contractile protein expression compared to wildtype cells. Ultracentrifugation assays after treating Acta2+/- R258C TGSMCs with phalloidin showed actin filament fractions, indicating that mutant α-actin can polymerize into filaments. Both Acta2+/+ R258C TGand Acta2+/- R258C TGSMCs have larger and more peripheral focal adhesions compared to wildtype SMCs. Rac1 was more activated in Acta2+/+ R258C TGSMCs; both Rac1 and RhoA were less activated in Acta2+/- R258C TG SMCs, and FAK was more activated in both transgenic SMC lines compared to wildtype. Proliferation in both cell lines was significantly increased compared to wildtype cells and could be partially attenuated by inhibition of FAK or PDGFRβ. These data support a dominant negative effect of the Acta2 R258C mutation on the SMC phenotype, with increasing phenotypic severity when wildtype: mutant α-actin levels are decreased.