Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

Degrees of macrophage-facilitated healing in aneurysm occlusion devices.

Insufficient healing of aneurysms following treatment with vascular occlusion devices put patients at severe risk of fatal rupture. Therefore, promoting healing and not just occlusion is vital to enhance aneurysm healing. Following occlusion device implantation, healing is primarily orchestrated by macrophage immune cells, ending with fibroblasts depositing collagen to stabilize the aneurysm neck and dome, preventing rupture. Several modified occlusion devices are available currently on-market. Previous in vivo work demonstrated that modifications of occlusion devices with a shape memory polymer foam had enhanced aneurysm healing outcomes. To better understand cellular response to occlusion devices and improve aneurysm occlusion device design variables, we developed an in vitro assay to isolate prominent interactions between devices and key healing players: macrophages and fibroblasts. We used THP-1 monocyte derived macrophages and human dermal fibroblasts in our cell culture models. Macrophages were allowed device contact with on-market competitor aneurysm occlusion devices for up to 96 h, to allow for any spontaneous device-driven macrophage activation. Macrophage secreted factors were captured in the culture media, in response to device-specific activation. Fibroblasts were then exposed to device-conditioned macrophage media (with secreted factors alone), to determine if there were any device-induced changes in collagen secretion. Our in vitro studies were designed to test the direct effect of devices on macrophage activation, and the indirect effect of devices on collagen secretion by fibroblasts to promote aneurysm healing and stabilization. Over 96 h, macrophages displayed significant migration toward and interaction with all tested devices. As compared to other devices, shape memory polymer foams (SMM, Shape Memory Medical) induced significant changes in gene expression indicating a shift toward an anti-inflammatory pro-healing M2-like phenotype. Similarly, macrophages in contact with SMM devices secreted more vascular endothelial growth factor (VEGF) compared with other devices. Macrophage conditioned media from SMM-contacted macrophages actively promoted fibroblast secretion of collagen, comparable to amounts observed with exogenous stimulation via VEGF supplementation. Our data indicate that SMM devices may promote good aneurysm healing outcomes, because collagen production is an essential step to ultimately stabilize an aneurysm.

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..

Liquid crystal elastomer based dynamic device for urethral support: Potential treatment for stress urinary incontinence.

Stress urinary incontinence (SUI) is characterized by the involuntary loss of urine due to increased intra-abdominal pressure during coughing, sneezing, or exercising. SUI affects 20-40% of the female population and is exacerbated by aging. Severe SUI is commonly treated with surgical implantation of an autologous or a synthetic sling underneath the urethra for support. These slings, however, are static, and their tension cannot be non-invasively adjusted, if needed, after implantation. This study reports the fabrication of a novel device based on liquid crystal elastomers (LCEs) capable of changing shape in response to temperature increase induced by transcutaneous IR light. The shape change of the LCE-based device was characterized in a scar tissue phantom model. An in vitro urinary tract model was designed to study the efficacy of the LCE-based device to support continence and adjust sling tension with IR illumination. Finally, the device was acutely implanted and tested for induced tension changes in female multiparous New Zealand white rabbits. The LCE device achieved 5.6% ± 1.1% actuation when embedded in an agar gel with an elastic modulus of 100 kPa. The corresponding device temperature was 44.9 °C ± 0.4 °C, and the surrounding agar temperature stayed at 42.1 °C ± 0.4 °C. Leaking time in the in vitro urinary tract model significantly decreased (p < 0.0001) when an LCE-based cuff was sutured around the model urethra from 5.2min ± 1min to 2min ±0.5min when the cuff was illuminated with IR light. Normalized leak point force (LPF) increased significantly (p = 0.01) with the implantation of an LCE-CB cuff around the bladder neck of multiparous rabbits. It decreased significantly (p = 0.023) when the device was actuated via IR light illumination. These results demonstrate that LCE material could be used to fabricate a dynamic device for treating SUI in women.

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.