Molecular Imaging of Oxygenation Changes during Immunotherapy in Combination with Paclitaxel in Triple Negative Breast Cancer.

Hypoxia is a common feature of the tumor microenvironment, including that of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with a high five-year mortality rate. Using [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) imaging, we aimed to monitor changes in response to immunotherapy (IMT) with chemotherapy in TNBC. TNBC-tumor-bearing mice received paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 and anti-cytotoxic T-lymphocyte 4. FMISO-PET imaging was performed on treatment days 0, 6, and 12. Max and mean standard uptake values (SUVmax and SUVmean, respectively), histological analyses, and flow cytometry results were compared. FMISO-PET imaging revealed differences in tumor biology between treatment groups prior to tumor volume changes. 4T1 responders showed SUVmean 1.6-fold lower (p = 0.02) and 1.8-fold lower (p = 0.02) than non-responders on days 6 and 12, respectively. E0771 responders showed SUVmean 3.6-fold lower (p = 0.001) and 2.7-fold lower (p = 0.03) than non-responders on days 6 and 12, respectively. Immunohistochemical analyses revealed IMT plus PTX decreased hypoxia and proliferation and increased vascularity compared to control. Combination IMT/PTX recovered the loss of CD4+ T-cells observed with single-agent therapies. PET imaging can provide timely, longitudinal data on the TNBC tumor microenvironment, specifically intratumoral hypoxia, predicting therapeutic response to IMT plus chemotherapy.

Preclinical PET Imaging of Granzyme B Shows Promotion of Immunological Response Following Combination Paclitaxel and Immune Checkpoint Inhibition in Triple Negative Breast Cancer.

Advancements in monitoring and predicting of patient-specific response of triple negative breast cancer (TNBC) to immunotherapy (IMT) with and without chemotherapy are needed. Using granzyme B-specific positron emission tomography (GZP-PET) imaging, we aimed to monitor changes in effector cell activation in response to IMT with chemotherapy in TNBC. TNBC mouse models received the paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 (anti-PD1) and anti-cytotoxic T-lymphocyte 4 (anti-CTLA4). GZP-PET imaging was performed on treatment days 0, 3, and 6. Mean standard uptake value (SUVmean), effector cell fractions, and SUV histograms were compared. Mice were sacrificed at early imaging timepoints for cytokine and histological analyses. GZP-PET imaging data revealed differences prior to tumor volume changes. By day six, responders had SUVmean ≥ 2.2-fold higher (p < 0.0037) and effector cell fractions ≥ 1.9-fold higher (p = 0.03) compared to non-responders. IMT/PTX resulted in a significantly different SUV distribution compared to control, indicating broader distribution of activated intratumoral T-cells. IMT/PTX resulted in significantly more necrotic tumor tissue and increased levels of IL-2, 4, and 12 compared to control. Results implicate immunogenic cell death through upregulation of key Th1/Th2 cytokines by IMT/PTX. Noninvasive PET imaging can provide data on the TNBC tumor microenvironment, specifically intratumoral effector cell activation, predicting response to IMT plus chemotherapy.

Imaging for Response Assessment in Cancer Clinical Trials.

The use of biomarkers is integral to the routine management of cancer patients, including diagnosis of disease, clinical staging and response to therapeutic intervention. Advanced imaging metrics with computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) are used to assess response during new drug development and in cancer research for predictive metrics of response. Key components and challenges to identifying an appropriate imaging biomarker are selection of integral vs integrated biomarkers, choosing an appropriate endpoint and modality, and standardization of the imaging biomarkers for cooperative and multicenter trials. Imaging biomarkers lean on the original proposed quantified metrics derived from imaging such as tumor size or longest dimension, with the most commonly implemented metrics in clinical trials coming from the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and then adapted versions such as immune-RECIST (iRECIST) and Positron Emission Tomography Response Criteria in Solid Tumors (PERCIST) for immunotherapy response and PET imaging, respectively. There have been many widely adopted biomarkers in clinical trials derived from MRI including metrics that describe cellularity and vascularity from diffusion-weighted (DW)-MRI apparent diffusion coefficient (ADC) and Dynamic Susceptibility Contrast (DSC) or dynamic contrast enhanced (DCE)-MRI (Ktrans, relative cerebral blood volume (rCBV)), respectively. Furthermore, Fluorodexoyglucose (FDG), fluorothymidine (FLT), and fluoromisonidazole (FMISO)-PET imaging, which describe molecular markers of glucose metabolism, proliferation and hypoxia have been implemented into various cancer types to assess therapeutic response to a wide variety of targeted- and chemotherapies. Recently, there have been many functional and molecular novel imaging biomarkers that are being developed that are rapidly being integrated into clinical trials (with anticipation of being implemented into clinical workflow in the future), such as artificial intelligence (AI) and machine learning computational strategies, antibody and peptide specific molecular imaging, and advanced diffusion MRI. These include prostate-specific membrane antigen (PSMA) and trastuzumab-PET, vascular tumor burden extracted from contrast-enhanced CT, diffusion kurtosis imaging, and CD8 or Granzyme B PET imaging. Further excitement surrounds theranostic procedures such as the combination of 68Ga/111In- and 177Lu-DOTATATE to use integral biomarkers to direct care and personalize therapy. However, there are many challenges in the implementation of imaging biomarkers that remains, including understand the accuracy, repeatability and reproducibility of both acquisition and analysis of these imaging biomarkers. Despite the challenges associated with the biological and technical validation of novel imaging biomarkers, a distinct roadmap has been created that is being implemented into many clinical trials to advance the development and implementation to create specific and sensitive novel imaging biomarkers of therapeutic response to continue to transform medical oncology.

Comparison of Panitumumab-IRDye800CW and 5-Aminolevulinic Acid to Provide Optical Contrast in a Model of Glioblastoma Multiforme.

Maximal safe resection of malignant tissue is associated with improved progression-free survival and better response to radiation and chemotherapy for patients with glioblastoma (GBM). 5-Aminolevulinic acid (5-ALA) is the current FDA-approved standard for intraoperative brain tumor visualization. Unfortunately, autofluorescence in diffuse areas and high fluorescence in dense tissues significantly limit discrimination at tumor margins. This study is the first to compare 5-ALA to an investigational new drug, panitumumab-IRDye800CW, in the same animal model. A patient-derived GBM xenograft model was established in 16 nude mice, which later received injections of 5-ALA, panitumumab-IRDye800CW, IRDye800CW, 5-ALA and IRDye800CW, or 5-ALA and panitumumab-IRDye800CW. Brains were prepared for multi-instrument fluorescence imaging, IHC, and quantitative analysis of tumor-to-background ratio (TBR) and tumor margin accuracy. Statistical analysis was compared with Wilcoxon rank-sum or paired t test. Panitumumab-IRDye800CW had a 30% higher comprehensive TBR compared with 5-ALA (P = 0.0079). SDs for core and margin regions of interest in 5-ALA-treated tissues were significantly higher than those found in panitumumab-IRDye800CW-treated tissues (P = 0.0240 and P = 0.0284, respectively). Panitumumab-IRDye800CW specificities for tumor core and margin were more than 10% higher than those of 5-ALA. Higher AUC for panitumumab-IRDye800CW indicated strong capability to discriminate between normal and malignant brain tissue when compared with 5-ALA. This work demonstrates that panitumumab-IRDye800CW shows potential as a targeting agent for fluorescence intraoperative detection of GBM. Improved margin definition and surgical resection using panitumumab-IRDye800 has the potential to improve surgical outcomes and survival in patients with GBM compared with 5-ALA.

Calmodulin antagonist enhances DR5-mediated apoptotic signaling in TRA-8 resistant triple negative breast cancer cells.

Patients with triple negative breast cancer (TNBC) have no successful "targeted" treatment modality, which represents a priority for novel therapy strategies. Upregulated death receptor 5 (DR5) expression levels in breast cancer cells compared to normal cells enable TRA-8, a DR5 specific agonistic antibody, to specifically target malignant cells for apoptosis without inducing normal hepatocyte apoptosis. Drug resistance is a common obstacle in TRAIL-based therapy for TNBC. Calmodulin (CaM) is overexpressed in breast cancer. In this study, we characterized the novel function of CaM antagonist in enhancing TRA-8 induced cytotoxicity in TRA-8 resistant TNBC cells and its underlying molecular mechanisms. Results demonstrated that CaM antagonist(s) enhanced TRA-8 induced cytotoxicity in a concentration and time-dependent manner for TRA-8 resistant TNBC cells. CaM directly bound to DR5 in a Ca2+ dependent manner, and CaM siRNA promoted DR5 recruitment of FADD and caspase-8 for DISC formation and TRA-8 activated caspase cleavage for apoptosis in TRA-8 resistant TNBC cells. CaM antagonist, trifluoperazine, enhanced TRA-8 activated DR5 oligomerization, DR5-mediated DISC formation, and TRA-8 activated caspase cleavage for apoptosis, and decreased anti-apoptotic pERK, pAKT, XIAP, and cIAP-1 expression in TRA-8 resistant TNBC cells. These results suggest that CaM could be a key regulator to mediate DR5-mediated apoptotic signaling, and suggests a potential strategy for using CaM antagonists to overcome drug resistance of TRAIL-based therapy for TRA-8 resistant TNBC.

Characterization of calmodulin-Fas death domain interaction: an integrated experimental and computational study.

The Fas death receptor-activated death-inducing signaling complex (DISC) regulates apoptosis in many normal and cancer cells. Qualitative biochemical experiments demonstrate that calmodulin (CaM) binds to the death domain of Fas. The interaction between CaM and Fas regulates Fas-mediated DISC formation. A quantitative understanding of the interaction between CaM and Fas is important for the optimal design of antagonists for CaM or Fas to regulate the CaM-Fas interaction, thus modulating Fas-mediated DISC formation and apoptosis. The V254N mutation of the Fas death domain (Fas DD) is analogous to an identified mutant allele of Fas in lpr-cg mice that have a deficiency in Fas-mediated apoptosis. In this study, the interactions of CaM with the Fas DD wild type (Fas DD WT) and with the Fas DD V254N mutant were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and molecular dynamics (MD) simulations. ITC results reveal an endothermic binding characteristic and an entropy-driven interaction of CaM with Fas DD WT or with Fas DD V254N. The Fas DD V254N mutation decreased the association constant (Ka) for CaM-Fas DD binding from (1.79 ± 0.20) × 10(6) to (0.88 ± 0.14) × 10(6) M(-1) and slightly increased a standard state Gibbs free energy (ΔG°) for CaM-Fas DD binding from -8.87 ± 0.07 to -8.43 ± 0.10 kcal/mol. CD secondary structure analysis and MD simulation results did not show significant secondary structural changes of the Fas DD caused by the V254N mutation. The conformational and dynamical motion analyses, the analyses of hydrogen bond formation within the CaM binding region, the contact numbers of each residue, and the electrostatic potential for the CaM binding region based on MD simulations demonstrated changes caused by the Fas DD V254N mutation. These changes caused by the Fas DD V254N mutation could affect the van der Waals interactions and electrostatic interactions between CaM and Fas DD, thereby affecting CaM-Fas DD interactions. Results from this study characterize CaM-Fas DD interactions in a quantitative way, providing structural and thermodynamic evidence of the role of the Fas DD V254N mutation in the CaM-Fas DD interaction. Furthermore, the results could help to identify novel strategies for regulating CaM-Fas DD interactions and Fas DD conformation and thus to modulate Fas-mediated DISC formation and thus Fas-mediated apoptosis.