Targeting myeloperoxidase limits myeloid cell immunosuppression enhancing immune checkpoint therapy for pancreatic cancer.

Pancreatic ductal adenocarcinoma is a devastating disease characterized by an extreme resistance to current therapies, including immune checkpoint therapy. The limited success of immunotherapies can be attributed to a highly immunosuppressive pancreatic cancer microenvironment characterized by an extensive infiltration of immune suppressing myeloid cells. While there are several pathways through which myeloid cells contribute to immunosuppression, one important mechanism is the increased production of reactive oxygen species. Here, we evaluated the contribution of myeloperoxidase, a myeloid-lineage restricted enzyme and primary source of reactive oxygen species, to regulate immune checkpoint therapy response in preclinical pancreatic cancer models. We compared treatment outcome, immune composition and characterized myeloid cells using wild-type, myeloperoxidase-deficient, and myeloperoxidase inhibitor treated wild-type mice using established subcutaneous pancreatic cancer models. Loss of host myeloperoxidase and pharmacological inhibition of myeloperoxidase in combination with immune checkpoint therapy significantly delayed tumor growth. The tumor microenvironment and systemic immune landscape demonstrated significant decreases in myeloid cells, exhausted T cells and T regulatory cell subsets when myeloperoxidase was deficient. Loss of myeloperoxidase in isolated myeloid cell subsets from tumor-bearing mice resulted in decreased reactive oxygen species production and T cell suppression. These data suggest that myeloperoxidase contributes to an immunosuppressive microenvironment and immune checkpoint therapy resistance where myeloperoxidase inhibitors have the potential to enhance immunotherapy response. Repurposing myeloperoxidase specific inhibitors may provide a promising therapeutic strategy to expand therapeutic options for pancreatic cancer patients to include immunotherapies.

Pancreatectomy Induces Cancer-Promoting Neutrophil Extracellular Traps.

BACKGROUND: Neutrophil extracellular traps (NETs) occur when neutrophil chromatin is decondensed and extruded into the extracellular space in a web-like structure. Originally described as an anti-microbial function, this process has been implicated in the pathogenesis of pancreatic disease. In addition, NETs are upregulated during physiologic wound-healing and coagulation. This study evaluated how the inflammatory response to pancreatic surgery influences NET formation. METHODS: For this study, 126 patients undergoing pancreatectomy gave consent before participation. Plasma was collected at several time points (preoperatively and through the postoperative outpatient visit). Plasma levels of NET markers, including cell-free DNA (cfDNA), citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-6, and granulocyte colony-stimulating factor (G-CSF) were measured using enzyme-linked immunosorbent assay (ELISA). Patient clinical data were retrospectively collected from a prospectively maintained database. RESULTS: After pancreatic resection, NET markers (cfDNA and CitH3) were elevated, peaking on postoperative days 3 and 4. This increase in NETs was due to an inherent change in neutrophil biology. Postoperatively, NET-inducing cytokines (IL-8, IL-6, and G-CSF) were increased, peaking early in the postoperative course. The patients undergoing the robotic approach had a reduction in NETs during the postoperative period compared with those who underwent the open approach. The patients who experienced a pancreatic leak had an increase in NET markers during the postoperative period. CONCLUSIONS: Pancreatectomy induces cancer-promoting NET formation. The minimally invasive robotic approach may induce fewer NETs, although the current analysis was limited by selection bias. Pancreatic leak resulted in increased NETs. Further study into the potential for NET inhibition during the perioperative period is warranted.

Development of a Potential Gallium-68-Labelled Radiotracer Based on DOTA-Curcumin for Colon-Rectal Carcinoma: From Synthesis to In Vivo Studies.

Colorectal cancer is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women worldwide. We have recently reported that curcuminoid complexes labelled with gallium-68 have demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compared to normal human lymphocytes. In the present study, we report a new gallium-68-labelled curcumin derivative (68Ga-DOTA-C21) and its initial validation as marker for early detection of colorectal cancer. The precursor and non-radioactive complexes were synthesized and deeply characterized by analytical methods then the curcuminoid was radiolabelled with gallium-68. The in vitro stability, cell uptake, internalization and efflux properties of the probe were studied in HT29 cells, and the in vivo targeting ability and biodistribution were investigated in mice bearing HT29 subcutaneous tumour model. 68Ga-DOTA-C21 exhibits decent stability (57 ± 3% after 120 min of incubation) in physiological media and a curcumin-mediated cellular accumulation in colorectal cancer cell line (121 ± 4 KBq of radiotracer per mg of protein within 60 min of incubation). In HT29 tumour-bearing mice, the tumour uptake of 68Ga-DOTA-C21 is 3.57 ± 0.3% of the injected dose per gram of tissue after 90 min post injection with a tumour to muscle ratio of 2.2 ± 0.2. High amount of activity (12.73 ± 1.9% ID/g) is recorded in blood and significant uptake of the radiotracer occurs in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). Further studies are needed to understand the mechanism of accumulation and clearance; however, 68Ga-DOTA-C21 provides a productive base-structure to develop further radiotracers for imaging of colorectal cancer.

An MRI-sensitive, non-photobleachable porphysome photothermal agent.

Photothermal therapy makes use of photothermal sensitizers and laser light to thermally ablate diseased tissues. Porphysome nanoparticles offer a nontoxic alternative to inorganic nanocrystals for the efficient conversion of light into heat. Mn(3+) ions were incorporated directly into the building blocks of our porphysome nanoparticles, thus imparting MRI sensitivity while simultaneously improving photostability and maintaining high photothermal efficiency. Mn porphysomes are as photothermally effective as free-base porphysomes and can rival gadolinium diethylenetriaminepentaacetate (Gd-DTPA) for MRI contrast generation. Their MRI contrast generation, photothermal efficiency, and photostability are unprecedented for an all-organic nanoparticle composed of a single functional component.

Group A streptococcal collagen-like protein 1 restricts tumor growth in murine pancreatic adenocarcinoma and inhibits cancer-promoting neutrophil extracellular traps.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. Methods: In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Mice harboring Panc02 or KPC subcutaneous tumors injected with three different M-type GAS strains. Tumors and spleens were harvested at the endpoint of the experiments to assess bacterial colonization and systemic spread, while sera were analyzed for humoral responses toward the streptococcal antigens, especially the M1 and Scl1 proteins. Role of the streptococcal collagen-like protein 1 (Scl1) in anti-PDAC activity was assessed in vivo after intratumoral injection with M1 GAS wild-type, an isogenic mutant strain devoid of Scl1, or a complemented mutant strain with restored scl1 expression. In addition, recombinant Scl1 proteins were tested for NET inhibition using in vitro and ex vivo assays assessing NET production and myeloperoxidase activity. Results: Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on Scl1, as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were weakly immunogenic toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Discussion: Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Group A Streptococcal Collagen-like Protein 1 Restricts Tumor Growth in Murine Pancreatic Adenocarcinoma and Inhibits Cancer-Promoting Neutrophil Extracellular Traps.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on streptococcal collagen-like protein 1 (Scl1), as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were negative toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Inhibition of myeloperoxidase enhances immune checkpoint therapy for melanoma.

BACKGROUND: The presence of a highly immunosuppressive tumor microenvironment has limited the success of immune checkpoint therapy (ICT). Immune suppressing myeloid cells with increased production of reactive oxygen species are critical drivers of this immunosuppressive tumor microenvironment. Strategies to limit these immune suppressing myeloid cells are needed to enhance response to ICT. METHODS: To evaluate the contribution of myeloperoxidase (MPO), a myeloid lineage-restricted enzyme and a major source of reactive oxygen species, to mediating ICT response, we compared treatment outcome and immune composition in wild-type, MPO-deficient (MPO -/- ), and MPO inhibitor-treated wild-type mice using established primary melanoma models. RESULTS: Tumor growth and survival studies demonstrated that either host deficiency (MPO -/- ) or pharmacological inhibition of MPO enhanced ICT response in two preclinical models of established primary melanoma in aged animals. The tumor microenvironment and systemic immune landscape underwent striking changes in infiltration of myeloid cells, T cells, B cells, and dendritic cells in MPO -/- mice; furthermore, a significant increase in myeloid cells was observed in ICT non-responders. The contribution of CD4+ T cells and NK cells during ICT response also changed in MPO -/- mice. Interestingly, MPO enzymatic activity, but not protein, was increased in CD11b+Ly6G+ myeloid cells isolated from marrow, spleen, and peritoneal cavities of mice bearing untreated melanoma, indicating systemic activation of innate immunity. Notably, repurposing MPO-specific inhibitors (verdiperstat, AZD5904) in combination with ICT pointedly enhanced response rates above ICT alone. Indeed, long-term survival was 100% in the YUMM3.3 melanoma model on treatment with verdiperstat plus ICT. CONCLUSION: MPO contributes to ICT resistance in established melanoma. Repurposing MPO-specific inhibitors may provide a promising therapeutic strategy to enhance ICT response.

Intrinsically copper-64-labeled organic nanoparticles as radiotracers.

PET friendly: labels for PET imaging are incorporated into completely organic porphysomes by using a fast (30 min), one-pot, high-yielding (>95 %) procedure to produce highly stable (>48 h) radiolabeled nanoparticles that show the highest specific activity ever reported for a (64) Cu-labeled nanoparticle. These (64) Cu-porphysomes can be accurately and noninvasively tracked in vivo.

Imaging of specific activation of photodynamic molecular beacons in breast cancer vertebral metastases.

Breast cancer is the second leading cause of cancer-related death in women. Approximately 85% of patients with advanced cases will develop spinal metastases. The vertebral column is the most common site of breast cancer metastases, where overexpression of matrix metalloproteinases (MMPs) promotes the spread of cancer. Current therapies have significant limitations due to the high associated risk of damaging the spinal cord. An attractive alternative is photodynamic therapy providing noninvasive and site-selective treatment. However, current photosensitizers are limited by their nonspecific accumulation. Photodynamic molecular beacons (PP(MMP)B), activated by MMPs, offer another level of PDT selectivity and image-guidance preserving criticial tissues, specifically the spinal cord. Metastatic human breast carcinoma cells, MT-1, were used to model the metastatic behavior of spinal lesions. In vitro and in vivo evidence demonstrates MMP specific activation of PP(MMP)B in MT-1 cells. Using a clinically relevant metastatic model, fluorescent imaging establishes the specific activation of PP(MMP)B by vertebral metastases versus normal tissue (i.e., spinal cord) demonstrating the specificity of these beacons. Here, we validate that the metastasis-selective mechanism of PP(MMP)Bs can specifically image breast cancer vertebral metastases, thereby differentiating tumor and healthy tissue.

Peptide-based molecular beacons for cancer imaging and therapy.

Peptide-based molecular beacons are Förster resonance energy transfer-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets and thus are useful tools for in vivo cancer imaging. With our increasing knowledge about human genome in health and disease, peptide-based "smart" probes are continually developed for in vivo optical imaging of specific molecular targets, biological pathways and cancer progression and diagnosis. A class of fluorescent photosensitizers further extends the application of peptide beacons to cancer therapeutics. This review highlights the applications of peptide beacons in cancer imaging, the simultaneous treatment and response monitoring and smart therapeutics with a focus on recent improvements in the design of these probes.

Multi-Modal Multi-Spectral Intravital Macroscopic Imaging of Signaling Dynamics in Real Time during Tumor-Immune Interactions.

A major obstacle in studying the interplay between cancer cells and the immune system has been the examination of proposed biological pathways and cell interactions in a dynamic, physiologically relevant system in vivo. Intravital imaging strategies are one of the few molecular imaging techniques that can follow biological processes at cellular resolution over long periods of time in the same individual. Bioluminescence imaging has become a standard preclinical in vivo optical imaging technique with ever-expanding versatility as a result of the development of new emission bioluminescent reporters, advances in genomic techniques, and technical improvements in bioluminescence imaging and processing methods. Herein, we describe an advance of technology with a molecular imaging window chamber platform that combines bioluminescent and fluorescent reporters with intravital macro-imaging techniques and bioluminescence spectral unmixing in real time applied to heterogeneous living systems in vivo for evaluating tumor signaling dynamics and immune cell enzyme activities concurrently.

Multi-Modal Multi-Spectral Intravital Microscopic Imaging of Signaling Dynamics in Real-Time during Tumor-ImmuneInteractions.

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

Myeloid cell-derived HOCl is a paracrine effector that trans-inhibits IKK/NF-κB in melanoma cells and limits early tumor progression.

The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity. Upon MDC activation, MPO is secreted into phagosomes where it catalyzes the production of hypochlorous acid (HOCl), a potent chlorinating oxidant. Here, we demonstrated that the myeloid lineage-restricted MPO-HOCl system had antitumor effects in early melanoma growth in aged mice. Orthotopic melanomas grew more slowly in immunocompetent MPO+/+ host mice compared to age-matched syngeneic MPO-/- mice. Real-time intravital tumor imaging in vivo and in cell cocultures revealed a cell-cell proximity-dependent association between MDC-derived MPO enzyme activity and blockade of ligand-induced IκBα degradation in tumor cells. HOCl directly trans-inhibited IκB kinase (IKK) activity in tumor cells, thereby decreasing nuclear factor κB (NF-κB) transcriptional activation and inducing changes in the expression of genes involved in metabolic pathways, cell cycle progression, and DNA replication. By contrast, HOCl induced transcriptional changes in CD8+ T cells related to ion transport and the MAPK and PI3K-AKT signaling pathways that are associated with T cell activation. MPO increased the circulating concentrations of the myeloid cell-attracting cytokines CXCL1 and CXCL5, enhanced local infiltration by CD8+ cytotoxic T cells, and decreased tumor growth. Overall, these data reveal a role for MDC-derived HOCl as a small-molecule paracrine signaling factor that trans-inhibits IKK in melanoma tumor cells, mediating antitumor responses during early tumor progression.

Interrogating Cellular Communication in Cancer with Genetically Encoded Imaging Reporters.

Cells continuously communicate changes in their microenvironment, both locally and globally, with other cells in the organism. Integration of information arising from signaling networks impart continuous, time-dependent changes of cell function and phenotype. Use of genetically encoded reporters enable researchers to noninvasively monitor time-dependent changes in intercellular and intracellular signaling, which can be interrogated by macroscopic and microscopic optical imaging, nuclear medicine imaging, MRI, and even photoacoustic imaging techniques. Reporters enable noninvasive monitoring of changes in cell-to-cell proximity, transcription, translation, protein folding, protein association, protein degradation, drug action, and second messengers in real time. Because of their positive impact on preclinical research, attempts to improve the sensitivity and specificity of these reporters, and to develop new types and classes of reporters, remain an active area of investigation. A few reporters have migrated to proof-of-principle clinical demonstrations, and recent advances in genome editing technologies may enable the use of reporters in the context of genome-wide analysis and the imaging of complex genomic regulation in vivo that cannot be readily investigated through standard methodologies. The combination of genetically encoded imaging reporters with continuous improvements in other molecular biology techniques may enhance and expedite target discovery and drug development for cancer interventions and treatment. © RSNA, 2020.

"Zipper" molecular beacons: a generalized strategy to optimize the performance of activatable protease probes.

We report the proof-of-principle concept for zipper molecular beacons (ZMB) comprising an asymmetrical polyarginine/polyglutamate electrostatic "zipper" hairpin-linked fluorophore-quencher pair. The objective is to balance maximal quenching efficiency and optimal two-step activation (protease cleavage/zipper dissociation), while enhancing target cell uptake. This strategy also eliminates the peptide sequence dependence of conventional protease beacons. This ZMB concept is a generalizable approach to improve the functionality of a wide range of diagnostic/therapeutic probes through a simple switching of substrate sequences.

Activation kinetics of zipper molecular beacons.

Proteases play key roles in the regulation of normal cellular function, and thus, their deregulation leads to many disease states. Molecular beacons are promising protease-imaging probes for the detection and characterization of disease as well as for the evaluation of treatment. Inspired by this, we examined the efficiency of zipper molecular beacons (ZMBs) as imaging probes. First, we showed experimentally that the symmetrical ZMB (zip5e5r), bearing 5-arginine and 5-glutamate arms, is as efficient as the asymmetrical zip5e8r in enhancing cell uptake but without the dark toxicity exhibited by the asymmetric zipper. Also, zip5e5r was shown to dissociate more efficiently at pH's greater than 5. Using a simple two-state binding model, we attributed this to a larger number of charge-pair conformations for zip5e8r. We then measured the ability of soluble matrix metalloproteinases (MMPs) to cleave zip5e5r, and compared their cleavage efficiency with the original photodynamic molecular beacon (PMB). Finally, as a first step toward understanding our observations quantitatively, we simulated the native structures of the peptides GPLGLARK and EGPLGLARRK with charged termini NH3(+) and COO(-) that approximate the PMB and ZMB (with one pair of arginine/glutamate electrostatic zipper), respectively. We concluded that inclusion of the zipper changes the native structure of the MBs, altering the cleavage efficiency of different MMPs.

Optical glucose analogs of aminolevulinic acid for fluorescence-guided tumor resection and photodynamic therapy.

PURPOSE: We have developed and tested a novel conjugation of the clinically used prodrug aminolevulinic acid with 2-deoxyglucosamine as a novel probe (ALA-2DG) for fluorescence imaging and photodynamic therapy. PROCEDURES: ALA-2DG was successfully synthesized, and the mechanisms of probe uptake, PpIX synthesis, and photodynamic therapy efficacy were evaluated in vitro and in vivo. RESULTS: ALA-2DG led to PpIX synthesis in tumor cells in vitro and in tumor in vivo. Competitive and inhibitory assays in vitro showed a reduction of this PpIX synthesis that was not observed when cells were incubated with ALA itself, indicating that intracellular uptake of ALA-2DG occurs by GLUT-mediated active transport. Initial photodynamic therapy studies confirmed the efficacy of ALA-2DG as a photodynamic sensitizer. CONCLUSIONS: The in vitro assays suggest that ALA-2DG is taken up by cells via glucose transporters. Initial studies in oral cancer demonstrated the applicability of ALA-2DG for in vivo imaging and its potential as an alternative to ALA-PpIX-based fluorescence diagnostics and photodynamic therapy, providing higher tumor specificity.

Inherently multimodal nanoparticle-driven tracking and real-time delineation of orthotopic prostate tumors and micrometastases.

Prostate cancer is the most common cancer among men and the second cause of male cancer-related deaths. There are currently three critical needs in prostate cancer imaging to personalize cancer treatment: (1) accurate intraprostatic imaging for multiple foci and extra-capsular extent; (2) monitoring local and systemic treatment response and predicting recurrence; and (3) more sensitive imaging of occult prostate cancer bone metastases. Recently, our lab developed porphysomes, inherently multimodal, all-organic nanoparticles with flexible and robust radiochemistry. Herein, we validate the first in vivo application of (64)Cu-porphysomes in clinically relevant orthotopic prostate and bony metastatic cancer models. We demonstrate clear multimodal delineation of orthotopic tumors on both the macro- and the microscopic scales (using both PET and fluorescence) and sensitively detected small bony metastases (<2 mm). The unique and multifaceted properties of porphysomes offers a promising all-in-one prostate cancer imaging agent for tumor detection and treatment response/recurrence monitoring using both radionuclide- and photonic-based strategies.

Biologically-targeted detection of primary and micro-metastatic ovarian cancer.

Ovarian cancer is the leading cause of morbidity/mortality from gynecologic malignancy. Early detection of disease is difficult due to the propensity for ovarian cancer to disseminate throughout the peritoneum. Currently, there is no single accurate test to detect primary or recurrent ovarian cancer. We report a novel clinical strategy using PPF: a multimodal, PET and optical, folate receptor (FR)-targeted agent for ovarian cancer imaging. The capabilities of PPF were evaluated in primary human ovarian cancer cells, in vivo xenografts derived from primary cells and ex vivo patient omemtum, as the heterogeneity and phenotype displayed by patients is retained. Primary cells uptake PPF in a FR-dependent manner demonstrating approximately a 5- to 25-fold increase in fluorescence. By both PET and fluorescence imaging, PPF specifically delineated FR-positive, ovarian cancer xenografts, with similar tumor-to-background ratios of 8.91±0.91 and 7.94±3.94, and micro-metastatic studding (<1mm), which demonstrated a 3.5-fold increase in PPF uptake over adjacent normal tissue. Ex vivo patient omentum demonstrated selective uptake of PFF by tumor deposits. The ability of PPF to identify metastatic deposits <1mm could facilitate more complete debulking (currently, optimal debulking is <10mm residual tumor), by providing a more sensitive imaging strategy improving treatment planning, response assessment and residual/recurrent disease detection. Therefore, PPF is a novel clinical imaging strategy that could substantially improve the prognosis of patients with ovarian cancer by allowing pre-, post- and intra-operative tumor monitoring, detection and possibly treatment throughout all stages of therapy and tumor progression.