High content, quantitative AFM analysis of the scalable biomechanical properties of extracellular vesicles.

Extracellular vesicles (EVs) are studied extensively as natural biomolecular shuttles and for their diagnostic and therapeutic potential. This exponential rise in interest has highlighted the need for highly robust and reproducible approaches for EV characterisation. Here we optimise quantitative nanomechanical tools and demonstrate the advantages of EV population screening by atomic force microscopy (AFM). Our high-content informatics analytical tools are made available for use by the EV community for widespread, standardised determination of structural stability. Ultracentrifugation (UC) and sonication, the common mechanical techniques used for EV isolation and loading respectively, are used to demonstrate the utility of optimised PeakForce-Quantitative Nano Mechanics (PF-QNM) analysis. EVs produced at an industrial scale exhibited biochemical and biomechanical alterations after exposure to these common techniques. UC resulted in slight increases in physical dimensions, and decreased EV adhesion concurrent with a decrease in CD63 content. Sonicated EVs exhibited significantly reduced levels of CD81, a decrease in size, increased Young's modulus and decreased adhesive force. These biomechanical and biochemical changes highlight the effect of EV sample preparation techniques on critical properties linked to EV cellular uptake and biological function. PF-QNM offers significant additional information about the structural information of EVs following their purification and downstream processing, and the analytical tools will ensure consistency of analysis of AFM data by the EV community, as this technique continues to become more widely implemented.

Vitamin D3 suppresses morphological evolution of the cribriform cancerous phenotype.

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3,theactive form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted "Swiss cheese-like" cribriform morphology (CM) comprising multiple abnormal "back to back" lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.

PI3'-kinase inhibition forestalls the onset of MEK1/2 inhibitor resistance in BRAF-mutated melanoma.

UNLABELLED: Phosphatidylinositide 3' (PI3')-lipid signaling cooperates with oncogenic BRAF(V600E) to promote melanomagenesis. Sustained PI3'-lipid production commonly occurs via silencing of the PI3'-lipid phosphatase PTEN or, less commonly, through mutational activation of PIK3CA, encoding the 110-kDa catalytic subunit of PI3'-kinase-α (PI3Kα). To define the PI3K catalytic isoform dependency of BRAF-mutated melanoma, we used pharmacologic, isoform-selective PI3K inhibitors in conjunction with melanoma-derived cell lines and genetically engineered mouse (GEM) models. Although BRAF(V600E)/PIK3CA(H1047R) melanomas were sensitive to the antiproliferative effects of selective PI3Kα blockade, inhibition of BRAF(V600E)/PTEN(Null) melanoma proliferation required combined blockade of PI3Kα, PI3Kδ, and PI3Kγ, and was insensitive to PI3Kß blockade. In GEM models, isoform-selective PI3K inhibition elicited cytostatic effects, but significantly potentiated melanoma regression in response to BRAF(V600E) pathway-targeted inhibition. Interestingly, PI3K inhibition forestalled the onset of MEK inhibitor resistance in two independent GEM models of BRAF(V600E)-driven melanoma. These results suggest that combination therapy with PI3K inhibitors may be a useful strategy to extend the duration of clinical response of patients with BRAF-mutated melanoma to BRAF(V600E) pathway-targeted therapies. SIGNIFICANCE: Although BRAF(V600E) pathway-targeted therapies elicit melanoma regression, the onset of drug resistance limits the durability of response. Here, we show that combined treatment with PI3K inhibitors significantly forestalled the onset of MEK1/2 inhibitor-resistant disease in BRAF-mutated GEM melanoma models. These results provide a conceptual framework for the combined deployment of BRAF(V600E) plus PI3K pathway-targeted inhibitors in the treatment of a subset of patients with BRAF-mutated melanoma.

Epithelial-specific loss of PTEN results in colorectal juvenile polyp formation and invasive cancer.

Cowden syndrome (CS) is a rare autosomal dominant cancer-prone disorder caused by germ-line mutation of the phosphatase and tensin homolog mutated on chromosome 10 (PTEN) tumor-suppressor gene. Affected patients commonly develop juvenile polyps, and show an elevated risk of developing colorectal cancers. The etiology of these peculiar polyps remains unclear, although previous work has suggested somatic PTEN alterations in the stroma of juvenile polyps. After a long latency period, we find epithelial-specific PTEN deletion to cause formation of juvenile polyps in the colorectum without stromal PTEN loss. More important, we find that these lesions closely recapitulate all of the characteristic histopathological features of juvenile polyps seen in patients with CS, including stromal alterations and dysplastic transformation to colorectal carcinoma. The stromal alterations we identify after epithelial-specific PTEN loss suggest that PTEN may be involved in altered epithelial-mesenchymal cross talk, which, in turn, predisposes to colorectal neoplasia and polyposis. Our transgenic model is the first to recapitulate colorectal juvenile polyposis in patients with CS. We conclude that stromal PTEN loss is not a prerequisite for the formation of juvenile polyps, and that colorectal juvenile polyps in CS are bona fide neoplastic precursor lesions.

PTEN loss and KRAS activation leads to the formation of serrated adenomas and metastatic carcinoma in the mouse intestine.

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co-occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal-specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild-type (WT) mice and mice with a predisposition to adenoma development (Apc(fl/+) ). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life-span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans.

Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma.

Malignant melanoma is frequently driven by mutational activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) accompanied by silencing of the phosphatase and tensin homology (PTEN) tumor suppressor. Despite the implied importance of PI3K signaling in PTENNull melanomas, mutational activation of the gene encoding the catalytic subunit of PI3Kα (PIK3CA), is rarely detected. Since PTEN has both PI3-lipid phosphatase-dependent and -independent tumor suppressor activities, we investigated the contribution of PI3K signaling to BRAFV600E-induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway-targeted inhibitors. These experiments revealed that mutationally activated PIK3CAH1047R cooperates with BRAFV600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAFV600E/PTENNull melanomas in vivo and in tissue culture. Combined inhibition of BRAFV600E and PI3K had more potent effects on the regression of established BRAFV600E/PTENNull melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAFV600E/PIK3CAH1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAFV600E/PTENNull melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAFV600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway-targeted inhibition of PI3K and BRAFV600E in the therapeutic management of BRAFV600E/PTENNull melanomas.