Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

Activation of Tumor-Cell STING Primes NK-Cell Therapy.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

Investigation of Kryptonite™ bone cement in hybrid screw configurations of locking plate humeral midshaft fixation: A study of surrogate bone model.

PURPOSE: Locking compression plates (LCP) allow trauma and orthopedic surgeons to have a variety of options for utilizing locking and non-locking screw features. In this study, the hybrid constructs of mixed unicortical and bicortical screws were investigated for humerus midshaft fractures. The locking and non-locking features were also incorporated into the hybrid LCP constructs. Kryptonite™ bone cement is biocompatible with low exothermic properties and strong metal adhesion. This novel bone cement was incorporated into the non-locking screw feature to compare its mechanical effect with that of a traditional locking screw feature. METHODS: A total of 24 synthetic bones (Sawbones(®) Inc., USA) were equally divided into three groups (n = 8). The control group obtained traditional LCP fixation (JSM Medimax Inc., India) with bicortical screws, while the hybrid locking constructs employed a mix of bicortical and unicortical screws. The two bicortical screws in hybrid constructs were placed at the end holes of the LCP constructs and the unicortical screws were placed into the remaining holes. The hybrid locking (HL) group contained locking unicortical screws, whereas the hybrid non-locking group (HNK) utilized non-locking screw features incorporating the Kryptonite™ bone cement in the plate/screw bone construct. Specimens were tested by dynamic and static analysis. The eight total constructs were equally divided into two subgroups (n = 4) to conduct axial compression and torsion test individually. The low cyclic test was conducted for dynamic analysis, which included 10,000 cycles at 1 Hz frequency with a cyclic loading of 0-500 N of axial force for the axial compression test and 0-20° of angular displacement for the torsion test. The static analysis was run by a failure test with a nondestructive strain rate of 0.1 mm/s for the axial compression test and 0.5° s(-1) for the torsional test. The construct stiffness of axial compression and torsion were derived from the linear portion of the load-displacement curves. The yield strength of axial compression and torsion was determined by using offset methods. The results of stiffness and yield strength were compared by using both one-way ANOVA and Scheffe and Games-Howell post hoc tests to analyze statically significant differences among the three groups. RESULTS: Specimens completed 10,000 cycles in the dynamic analysis of axial and torsional cyclic tests without major deformation. To compare with the control group in static analysis, the HL and HNK groups achieved positive effect in axial stiffness, 12.3% and 10.5% greater than the control group respectively. HL obtained axial yield strength about 12% less than the control group. The HNK group was equivalent with the control group in axial yield strength. The torsional stiffness and yield strength were found similar in three groups, which indicated torsional equivalence among them. The stiffness and yield strength from axial compression and torsion were found statically non-significant among three groups. CONCLUSIONS: Hybrid LCP constructs were initially showed to maintain equivalent axial and torsional stability with the traditional method. Kryptonite™ hosted mechanical and biological advantages for internal plate/screws (PLT) fixation.