Molecular portraits of cell cycle checkpoint kinases in cancer evolution, progression, and treatment responsiveness.

Cell cycle dysregulation is prerequisite for cancer formation. However, it is unknown whether the mode of dysregulation affects disease characteristics. Here, we conduct comprehensive analyses of cell cycle checkpoint dysregulation using patient data and experimental investigations. We find that ATM mutation predisposes the diagnosis of primary estrogen receptor (ER)+/human epidermal growth factor (HER)2- cancer in older women. Conversely, CHK2 dysregulation induces formation of metastatic, premenopausal ER+/HER2- breast cancer (P = 0.001) that is treatment-resistant (HR = 6.15, P = 0.01). Lastly, while mutations in ATR alone are rare, ATR/TP53 co-mutation is 12-fold enriched over expected in ER+/HER2- disease (P = 0.002) and associates with metastatic progression (HR = 2.01, P = 0.006). Concordantly, ATR dysregulation induces metastatic phenotypes in TP53 mutant, not wild-type, cells. Overall, we identify mode of cell cycle dysregulation as a distinct event that determines subtype, metastatic potential, and treatment responsiveness, providing rationale for reconsidering diagnostic classification through the lens of the mode of cell cycle dysregulation..

Decellularized organ biomatrices facilitate quantifiable in vitro 3D cancer metastasis models.

Metastatic cancers are chemoresistant, involving complex interplay between disseminated cancer cell aggregates and the distant organ microenvironment (extracellular matrix and stromal cells). Conventional metastasis surrogates (scratch/wound healing, Transwell migration assays) lack 3D architecture and ECM presence. Metastasis studies can therefore significantly benefit from biomimetic 3D in vitro models recapitulating the complex cascade of distant organ invasion and colonization by collective clusters of cells. We aimed to engineer reproducible and quantifiable 3D models of highly therapy-resistant cancer processes: (i) colorectal cancer liver metastasis; and (ii) breast cancer lung metastasis. Metastatic seeds are engineered using 3D tumor spheroids to recapitulate the 3D aggregation of cancer cells both in the tumor and in circulation throughout the metastatic cascade of many cancers. Metastatic soil was engineered by decellularizing porcine livers and lungs to generate biomatrix scaffolds, followed by extensive materials characterization. HCT116 colorectal and MDA-MB-231 breast cancer spheroids were generated on hanging drop arrays to initiate clustered metastatic seeding into liver and lung biomatrix scaffolds, respectively. Between days 3-7, biomatrix cellular colonization was apparent with increased metabolic activity and the presence of cellular nests evaluated via multiphoton microscopy. HCT116 and MDA-MB-231 cells colonized liver and lung biomatrices, and at least 15% of the cells invaded more than 20 µm from the surface. Engineered metastases also expressed increased signatures of genes associated with the metastatic epithelial to mesenchymal transition (EMT). Importantly, inhibition of matrix metalloproteinase-9 inhibited metastatic invasion into the biomatrix. Furthermore, metastatic nests were significantly more chemoresistant (>3 times) to the anti-cancer drug oxaliplatin, compared to 3D spheroids. Together, our data indicated that HCT116 and MDA-MB-231 spheroids invade, colonize, and proliferate in livers and lungs establishing metastatic nests in 3D settings in vitro. The metastatic nature of these cells was confirmed with functional readouts regarding EMT and chemoresistance. Modeling the dynamic metastatic cascade in vitro has potential to identify therapeutic targets to treat or prevent metastatic progression in chemoresistant metastatic cancers.

Macrophage activation in response to shape memory polymer foam-coated aneurysm occlusion devices.

Brain aneurysms can be treated with embolic coils using minimally invasive approaches. It is advantageous to modulate the biologic response of platinum embolic coils. Our previous studies demonstrated that shape memory polymer (SMP) foam coated embolization coils (FCC) devices demonstrate enhanced healing responses in animal models compared with standard bare platinum coil (BPC) devices. Macrophages are the most prevalent immune cell type that coordinate the greater immune response to implanted materials. Hence, we hypothesized that the highly porous SMP foam coatings on embolic coils activate a pro-regenerative healing phenotype. To test this hypothesis, we analyzed the number and type of infiltrating macrophages in FCC or BPC devices implanted in a rabbit elastase aneurysm model. FCC devices elicited a great number of infiltration macrophages, skewed significantly to a pro-regenerative M2-like phenotype 90 days following implantation. We devised an in vitro assay, where monocyte-derived macrophages were placed in close association with FCC or BPC devices for 6-72 h. Macrophages encountering SMP FCC-devices demonstrated highly mixed activation phenotypes at 6 h, heavily skewing toward an M2-like phenotype by 72 h, compared with macrophages encountering BPC devices. Macrophage activation was evaluated using gene expression analysis, and secreted cytokine evaluation. Together, our results demonstrate that FCC devices promoted a pro-regenerative macrophage activation phenotype, compared with BPC devices. Our in vitro findings corroborate with in vivo observations that SMP-based modification of embolic coils can promote better healing of the aneurysm site, by sustaining a pro-healing macrophage phenotype.