Influence of a metaphyseal sleeve on the stress-strain state of a bone-tumor implant system in the distal femur: an experimental and finite element analysis

Background Aseptic loosening of distal femoral tumor implants significantly correlates with the resection length. We designed a new “sleeve” that is specially engaged in the metaphysis at least 5 cm proximal to the knee joint line to preserve as much bone stock as possible. This study investigates the influence of a metaphyseal sleeve on the stress-strain state of a bone tumor implant system in the distal femur. Methods Cortex strains in intact and implanted femurs were predicted with finite element (FE) models. Moreover strains were experimentally measured in a cadaveric femur with and without a sleeve and stem under an axial compressive load of 1000 N. The FE models, which were validated by linear regression, were used to investigate the maximal von Mises stress and the implanted-to-intact (ITI) ratios of strain in the femur with single-legged stance loading under immediate postoperative and osseointegration conditions. Results Good agreement was noted between the experimental measurements and numerical predictions of the femoral strains (coefficient of determination (R2) ≥ 0.95; root-mean-square error (RMSE%) ≈ 10%). The ITI ratios for the metaphysis were between 13 and 28% and between 10 and 21% under the immediate postoperative and osseointegration conditions, respectively, while the ITI ratios for the posterior and lateral cortices around the tip of the stem were 110% and 119% under the immediate-postoperative condition, respectively, and 114% and 101% under the osseointegration condition, respectively. The maximal von Mises stresses for the implanted femur were 113.8 MPa and 43.41 MPa under the immediate postoperative and osseointegration conditions, which were 284% and 47% higher than those in the intact femur (29.6 MPa), respectively. Conclusions This study reveals that a metaphyseal sleeve may cause stress shielding relative to the intact femur, especially in the distal metaphysis. Stress concentrations might mainly occur in the posterior cortex around the tip of the stem. However, stress concentrations may not be accompanied by periprosthetic fracture under the single-legged stance condition.

141 PBMC-based cancer vaccines generated with microfluidics squeezing demonstrate synergistic and durable tumor reduction in combination with PD1 checkpoint and FAP targeted IL-2 variants

BackgroundWe engineered unfractionated peripheral blood mononuclear cells (PBMCs) to function as antigen presenting cells (APCs) that generate potent CD8+ T cell responses. We investigated the combined efficacy of PBMC-based cancer vaccine with targeted interleukin 2 variants (IL2v); anti-Programmed Cell Death Protein 1 (muPD1-IL2v) and anti-Fibroblast Activation Protein (muFAP-IL2v).MethodsWe generated PBMC-based cancer vaccine with microfluidic cell engineering system (Cell Squeeze®), which facilitates direct cytosolic antigen delivery and enables cell subsets within PBMCs to function as APCs. The immunocytokines used contain IL2v fused with antibody counterparts that enable targeting to tumor-associated stroma or immune cells (aFAP and aPD-1, respectively) with modified FcR binding. The IL2v moiety, compared with wild-type IL-2, has abolished binding to IL-2Ra (CD25) resulting in IL-2Rgb binding only, thus fully maintaining activity on NK and CD8+ T cells, while avoiding Treg activity and CD25 mediated toxicity.ResultsIn the murine TC-1 HPV tumor model, SQZ-PBMC-based vaccines show efficacy as monotherapy (1e6 cells administered iv on day 14 post-tumor implant), while SQZ combination therapy with targeted immunocytokines, muPD1-IL2v and muFAP-IL2v (2 mg/kg or 1 mg/kg, respectively, administered iv on days 21, 28, and 35 post-tumor implant) significantly delayed tumor growth and improved survival in murine TC-1 HPV tumor model. Median survival of combination treated groups remained undefined at day 84 post-tumor implant, while the monotherapy treated groups had calculated median survival times of 36.5, 42, and 70 days for the muFAP-IL2v, muPD1-IL2v, and SQZ monotherapy groups, respectively. Following initial tumor clearance, tumor-free mice (2/12 animals for SQZ monotherapy; 8/12 animals for SQZ with muFAP-IL2v; 11/11 animals for SQZ with muPD1-IL2v) were all re-challenged at day 84 and all remained tumor free at least 7 weeks post re-challenge, suggesting the generation of anti-tumor memory response. In a mechanistic study, SQZ-PBMCs in combination with muPD1-IL2v resulted in increased expansion of intra-tumoral, antigen-specific CD8+ T cells compared with separate administration of either therapy (~3.6-fold over SQZ alone; ~2000-fold over muPD1-IL2v alone; per mg of tumor). Combination therapy also resulted in improved IFNγ and TNFα cytokine production by SQZ-elicited CD8+ T cells (~1.7-fold and ~9-fold, respectively, over SQZ monotherapy).ConclusionsMonotherapy with SQZ-PBMC-based cancer vaccines can drive anti-tumor responses in murine systems. These responses are enhanced by combined administration of targeted immunocytokines. Monotherapy with SQZ-PBMC-HPV is currently under clinical evaluation for HPV16+ tumor indications. These preclinical data support the combination of SQZ-PBMC with FAP-IL2v or PD1-IL2v targeted immunocytokine as promising cancer immunotherapies.

TMOD-10. EFFECT OF BRAIN TUMORIGENESIS ON CEREBRAL CORTICAL FUNCTIONAL CONNECTIVITY IN THE MOUSE

INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bears pertinence to the pathogenesis of neuro-cognitive decline and tumor-associated epilepsy.

PATH-02. CHARACTERIZATION OF FUNCTIONAL NETWORK EFFECTS IN THE CEREBRAL CORTEX DURING BRAIN TUMORIGENESIS IN THE MOUSE

INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ flux imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bear pertinence to the pathogenesis of neuro-cognitive decline and tumor associated epilepsy.

TMIC-33. INVESTIGATING S1PR3-MEDIATED GLIOBLASTOMA INVASION MECHANISMS USING 3D HYDROGEL - TISSUE CULTURE INSERT MODEL

Glioblastoma (GBM) is the most common malignant brain tumor and is characterized by its ability to invade into the surrounding microenvironment of the brain. The invasiveness of GBM makes this cancer extremely hard to treat, leading to a median patient survival of less than 16 months. Interactions between the tumor and surrounding tumor microenvironment (TME) play a key role in glioma invasion. Previous data from our xenograft mouse tumor implant model displays increased invasion in regions of fluid flow. Using this model, we identified an upregulation of sphingosine-1-phosphate receptor 3 (S1PR3) in the TME in regions of flow. We used a syngeneic GL261 mouse model and found S1PR3-/- mice display decreased flow-mediated glioma invasion in comparison to wild type mice. To further understand the individual contributions of the S1PR3-presenting cells in the TME, we have examined the role of S1PR3 in our in vitro system. This system is based on patient derived cellular ratios and incorporates collagen-hyaluronan hydrogels placed within 96 well tissue culture insert plates. The tunability of this model allows for interactions between various cell types and the impact of fluid flow on invasion to be examined. To examine the role of S1PR3 on invasion, TY52156 (an S1PR3 inhibitor) was applied to different cellular combinations including: G34 alone (a patient-specific cell line), +astrocytes, +microglia, +TME (astrocytes and microglia). A significant decrease in G34 flow stimulated invasion was observed with TY52156 but only in the presence of the TME or microglia alone. This data suggests that TY52156 thwarts the effects of flow and the microglia contributions to invasion. To further this work we plan to identify and evaluate other S1PR3-expressing cell types from mouse tumor implant samples using immunohistochemistry staining. This information will be used to determine further components that can be examined in our in vitro model.