Structural and Mechanical Improvements to Bone Are Strain Dependent with Axial Compression of the Tibia in Female C57BL/6 Mice

Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function

Load/Strain Calibration Curve.

<p>This figure demonstrates the linear relationship between applied load and resulting tensile strain at the periosteal mid-diaphysis. For all data points, error bars represent the standard deviation from n = 5 mice.</p

Timeline for tibial loading study and waveform profile.

<p>A) The right tibia of each mouse was loaded using the waveform to a set force (8.8N, 10.6N and 12.4N) to elicit a desired periosteal mid-diaphyseal tensile strain level (1700 με, 2050 με and 2400 με) over a 14 day period. B) The loading profile consisted of four haversine waveforms followed by 3 seconds of rest repeated 55 times for a total of 220 cycles of loading per day.</p

Schematic Representations of Standard Site Geometric Profiles.

<p>A) 1700 με group. B) 2050 με group. C) 2400 με group. There was a dose-dependent increase in cortical parameters with robust periosteal and endocortical formation, especially at the higher strain levels. D) 2400 με group with the addition of those animals that experiences a woven bone response due to loading.</p

Schematic representations of mechanical testing curves.

<p>A) Representative structural-level force/displacement curves. B) Estimated tissue-level mechanical curves. At 1700 με, there was no mechanical effect noted. Those limbs loaded to 2050 με experienced significant increases in structural- and tissue-level strength and energy dissipation. The 2400 με group also experienced gains. However, when animals that experienced a woven bone response were removed from the analysis, the gains were more modest (especially at the tissue-level) and most failed to reach significance versus the contralateral control limb given the loss in power. For all data points, error bars represent the standard error of the mean (SEM).</p

PD-L1 expression and prognostic impact in glioblastoma

BACKGROUND: Therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and PD-1/PD-L1 has demonstrated tumor regression in clinical trials, and phase 2 trials are ongoing in glioblastoma (GBM). Previous reports have suggested that responses are more frequent in patients with tumors that express PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact. METHODS: Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with GBM. We categorized our results according to the total number of PD-L1-expressing cells within the GBMs and then validated this finding in ex vivo GBM flow cytometry with further analysis of the T cell populations. We then evaluated the association between PD-L1 expression and median survival time using the protein expression datasets and mRNA from The Cancer Genome Atlas. RESULTS: The median percentage of PD-L1-expressing cells in GBM by cell surface staining is 2.77% (range: 0%–86.6%; n = 92), which is similar to the percentage found by ex vivo flow cytometry. The majority of GBM patients (61%) had tumors with at least 1% or more PD-L1-positive cells, and 38% had at least 5% or greater PD-L1 expression. PD-L1 is commonly expressed on the GBM-infiltrating T cells. Expression of both PD-L1 and PD-1 are negative prognosticators for GBM outcome. CONCLUSIONS: The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome

Genomic Correlates of Outcome in Tumor-Infiltrating Lymphocyte Therapy for Metastatic Melanoma.

PURPOSE: Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TIL) historically yields a 40-50% response rate in metastatic melanoma. However, the determinants of outcome are largely unknown. EXPERIMENTAL DESIGN: We investigated tumor-based genomic correlates of overall survival (OS), progression-free survival (PFS), and response to therapy by interrogating tumor samples initially collected to generate TIL infusion products. RESULTS: Whole exome sequencing (WES) data from 64 samples indicated a positive correlation between neoantigen load and OS, but not PFS or response to therapy. RNA sequencing analysis of 34 samples showed that expression of PDE1C, RTKN2, and NGFR were enriched in responders who had improved PFS and OS. In contrast, the expression of ELFN1 was enriched in patients with unfavorable response, poor PFS and OS, whereas enhanced methylation of ELFN1 was observed in patients with favorable outcomes. Expression of ELFN1, NGFR and PDE1C was mainly found in cancer-associated fibroblasts and endothelial cells in tumor tissues across different cancer types in publicly available single cell RNA sequencing datasets, suggesting a role for elements of the tumor microenvironment in defining the outcome of TIL therapy. CONCLUSIONS: Our findings suggest that transcriptional features of melanomas correlate with outcomes after TIL therapy and may provide candidates to guide patient selection

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field