Cancer insights from magnetic resonance spectroscopy of cells and excised tumors.

Multinuclear ex vivo magnetic resonance spectroscopy (MRS) of cancer cells, xenografts, human cancer tissue, and biofluids is a rapidly expanding field that is providing unique insights into cancer. Starting from the 1970s, the field has continued to evolve as a stand-alone technology or as a complement to in vivo MRS to characterize the metabolome of cancer cells, cancer-associated stromal cells, immune cells, tumors, biofluids and, more recently, changes in the metabolome of organs induced by cancers. Here, we review some of the insights into cancer obtained with ex vivo MRS and provide a perspective of future directions. Ex vivo MRS of cells and tumors provides opportunities to understand the role of metabolism in cancer immune surveillance and immunotherapy. With advances in computational capabilities, the integration of artificial intelligence to identify differences in multinuclear spectral patterns, especially in easily accessible biofluids, is providing exciting advances in detection and monitoring response to treatment. Metabolotheranostics to target cancers and to normalize metabolic changes in organs induced by cancers to prevent cancer-induced morbidity are other areas of future development.

Twist alters the breast tumor microenvironment via choline kinase to facilitate an aggressive phenotype.

Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype.

Molecular and functional imaging insights into the role of hypoxia in cancer aggression.

Hypoxia in cancers has evoked significant interest since 1955 when Thomlinson and Gray postulated the presence of hypoxia in human lung cancers, based on the observation of necrosis occurring at the diffusion limit of oxygen from the nearest blood vessel, and identified the implication of these observations for radiation therapy. Coupled with discoveries in 1953 by Gray and others that anoxic cells were resistant to radiation damage, these observations have led to an entire field of research focused on exploiting oxygenation and hypoxia to improve the outcome of radiation therapy. Almost 65 years later, tumor heterogeneity of nearly every parameter measured including tumor oxygenation, and the dynamic landscape of cancers and their microenvironments are clearly evident, providing a strong rationale for cancer personalized medicine. Since hypoxia is a major cause of extracellular acidosis in tumors, here, we have focused on the applications of imaging to understand the effects of hypoxia in tumors and to target hypoxia in theranostic strategies. Molecular and functional imaging have critically important roles to play in personalized medicine through the detection of hypoxia, both spatially and temporally, and by providing new understanding of the role of hypoxia in cancer aggressiveness. With the discovery of the hypoxia-inducible factor (HIF), the intervening years have also seen significant progress in understanding the transcriptional regulation of hypoxia-induced genes. These advances have provided the ability to silence HIF and understand the associated molecular and functional consequences to expand our understanding of hypoxia and its role in cancer aggressiveness. Most recently, the development of hypoxia-based theranostic strategies that combine detection and therapy are further establishing imaging-based treatment strategies for precision medicine of cancer.

MRI and MRS of intact perfused cancer cell metabolism, invasion, and stromal cell interactions.

Because of the spatial and temporal heterogeneities of cancers, technologies to investigate cancer cells and the consequences of their interactions with abnormal physiological environments, such as hypoxia and acidic extracellular pH, with stromal cells, and with the extracellular matrix, under controlled conditions, are valuable to gain insights into the functioning of cancers. These insights can lead to an understanding of why cancers invade and metastasize, and identify effective treatment strategies. Here we have provided an overview of the applications of MRI/MRS/MRSI to investigate intact perfused cancer cells, their metabolism and invasion, and their interactions with stromal cells and the extracellular matrix.

A novel multiple biomarker panel for the early detection of high-grade serous ovarian carcinoma.

INTRODUCTION: Since the majority of patients are diagnosed at an advanced stage, ovarian cancer remains the most lethal gynecologic malignancy. There is no single biomarker with the sensitivity and specificity required for effective cancer screening; therefore, we investigated a panel of novel biomarkers for the early detection of high-grade serous ovarian carcinoma. METHODS: Twelve serum biomarkers with high differential gene expression and validated antibodies were selected: IL-1Ra, IL-6, Dkk-1, uPA, E-CAD, ErbB2, SLPI, HE4, CA125, LCN2, MSLN, and OPN. They were tested using Simple Plex™, a multi-analyte immunoassay platform, in samples collected from 172 patients who were either healthy, had benign gynecologic pathologies, or had high-grade serous ovarian adenocarcinomas. The receiver operating characteristic (ROC) curve, ROC area under the curve (AUC), and standard error (SE) of the AUC were obtained. Univariate ROC analyses and multivariate ROC analyses with the combination of multiple biomarkers were performed. RESULTS: The 4-marker panel consisting of CA125, HE4, E-CAD, and IL-6 had the highest ROC AUC. When evaluated for the ability to distinguish early stage ovarian cancer from a non-cancer control, not only did this 4-marker panel (AUC=0.961) performed better than CA 125 alone (AUC=0.851; P=0.0150) and HE4 alone (AUC=0.870; P=0.0220), but also performed significantly better than the 2- marker combination of CA125+HE4 (AUC=0.922; P=0.0278). The 4-marker panel had the highest average sensitivity under the region of its ROC curve corresponding to specificity ranging from 100% down to ~95%. CONCLUSION: The four-marker panel, CA125, HE4, E-CAD, and IL-6, shows potential in detecting serous ovarian cancer at earlier stages. Additional validation studies using the biomarker combination in ovarian cancer patients are warranted.

The HOXB7 protein renders breast cancer cells resistant to tamoxifen through activation of the EGFR pathway.

Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.

MR - eyes for cancer: looking within an impenetrable disease.

Probe development is a critical component in cancer imaging, and novel probes are making major inroads in several aspects of cancer detection and image-guided treatments. Intrinsic MR probes such as signals from metabolites and their chemical shifts have been used for more than a decade to understand cancer physiology and metabolism. Through the integration of technology, molecular biology, and chemistry, the last few years have witnessed an explosion of extrinsic probes for molecular and functional imaging of cancer that, together with techniques such as CEST and hyperpolarization, have significantly expanded the repertoire of MR techniques in basic and translational investigations of many different aspects of cancer. Furthermore, incorporation of MR probes into multifunctional nanoparticles and multimodality imaging platforms have opened new opportunities for MR in image-guided diagnosis and therapy of cancer. Here we have provided an overview of recent innovations that have occurred in the development of MRI probes for molecular and functional imaging of cancer. Although most of these novel probes are not clinically available, they offer significant promise for future translational applications. In this review, we have highlighted the areas of future development that are likely to have a profound impact on cancer detection and treatment.

PET/MRI and Bioluminescent Imaging Identify Hypoxia as a Cause of Programmed Cell Death Ligand 1 Image Heterogeneity.

Purpose To examine the association between hypoxia and programmed cell death ligand 1 (PD-L1) expression using bioluminescence imaging (BLI) and PET/MRI in a syngeneic mouse model of triple-negative breast cancer (TNBC). Materials and Methods PET/MRI and optical imaging were used to determine the role of hypoxia in altering PD-L1 expression using a syngeneic TNBC model engineered to express luciferase under hypoxia. Results Imaging showed a close spatial association between areas of hypoxia and increased PD-L1 expression in the syngeneic murine (4T1) tumor model. Mouse and human TNBC cells exposed to hypoxia exhibited a significant increase in PD-L1 expression, consistent with the in vivo imaging data. The role of hypoxia in increasing PD-L1 expression was further confirmed by using The Cancer Genome Atlas analyses of different human TNBCs. Conclusion These results have identified the potential role of hypoxia in contributing to PD-L1 heterogeneity in tumors by increasing cancer cell PD-L1 expression. Keywords: Hypoxia, PD-L1, Triple-Negative Breast Cancer, PET/MRI, Bioluminescence Imaging Supplemental material is available for this article. © RSNA, 2023.

Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice.

(1) Background: MDSCs play an active role in the immune surveillance escape of cancer cells. Because MDSCs in mice are CD11b+Gr1+, near-infrared photoimmunotherapy (NIR-PIT) using the NIR dye IR700 conjugated to an MDSC-binding antibody provides an opportunity for targeted elimination of MDSCs. (2) Methods: The efficacy of Gr1-IR700-mediated NIR-PIT was evaluated in vitro using magnetically separated CD11b+Gr1+ MDSCs from spleens of 4T1-luc tumor-bearing (TB) mice. For in vivo evaluation, spleens of Gr1-IR700-injected 4T1-luc TB mice were irradiated with NIR light, and splenocyte viability was determined using CCK-8 assays. Metabolic profiling of NIR-PIT-irradiated spleens was performed using 1H MRS. (3) Results: Flow cytometric analysis confirmed a ten-fold increase in splenic MDSCs in 4T1-luc TB mice. Gr1-IR700-mediated NIR-PIT eliminated tumor-induced splenic MDSCs in culture. Ex vivo fluorescence imaging revealed an 8- and 9-fold increase in mean fluorescence intensity (MFI) in the spleen and lungs of Gr1-IR700-injected compared to IgG-IR700-injected TB mice. Splenocytes from Gr1-IR700-injected TB mice exposed in vivo to NIR-PIT demonstrated significantly lower viability compared to no light exposure or untreated control groups. Significant metabolic changes were observed in spleens following NIR-PIT. (4) Conclusions: Our data confirm the ability of NIR-PIT to eliminate splenic MDSCs, identifying its potential to eliminate MDSCs in tumors to reduce immune suppression. The metabolic changes observed may identify potential biomarkers of splenic MDSC depletion as well as potential metabolic targets of MDSCs.

VEGF Overexpression Significantly Increases Nanoparticle-Mediated siRNA Delivery and Target-Gene Downregulation.

The availability of nanoparticles (NPs) to deliver small interfering RNA (siRNA) has significantly expanded the specificity and range of 'druggable' targets for precision medicine in cancer. This is especially important for cancers such as triple negative breast cancer (TNBC) for which there are no targeted treatments. Our purpose here was to understand the role of tumor vasculature and vascular endothelial growth factor (VEGF) overexpression in a TNBC xenograft in improving the delivery and function of siRNA NPs using in vivo as well as ex vivo imaging. We used triple negative MDA-MB-231 human breast cancer xenografts derived from cells engineered to overexpress VEGF to understand the role of VEGF and vascularization in NP delivery and function. We used polyethylene glycol (PEG) conjugated polyethylenimine (PEI) NPs to deliver siRNA that downregulates choline kinase alpha (Chkα), an enzyme that is associated with malignant transformation and tumor progression. Because Chkα converts choline to phosphocholine, effective delivery of Chkα siRNA NPs resulted in functional changes of a significant decrease in phosphocholine and total choline that was detected with 1H magnetic resonance spectroscopy (MRS). We observed a significant increase in NP delivery and a significant decrease in Chkα and phosphocholine in VEGF overexpressing xenografts. Our results demonstrated the importance of tumor vascularization in achieving effective siRNA delivery and downregulation of the target gene Chkα and its function.

PD-L1 near Infrared Photoimmunotherapy of Ovarian Cancer Model.

(1) Background: Despite advances in surgical approaches and drug development, ovarian cancer is still a leading cause of death from gynecological malignancies. Patients diagnosed with late-stage disease are treated with aggressive surgical resection and chemotherapy, but recurrence with resistant disease is often observed following treatment. There is a critical need for effective therapy for late-stage ovarian cancer. Photoimmunotherapy (PIT), using an antibody conjugated to a near infrared (NIR) dye, constitutes an effective theranostic strategy to detect and selectively eliminate targeted cell populations. (2) Methods: Here, we are targeting program death ligand 1 (PD-L1) using NIR-PIT in a syngeneic mouse model of ovarian cancer. PD-L1 PIT-mediated cytotoxicity was quantified in RAW264.7 macrophages and ID8-Defb29-VEGF cells in culture, and in vivo with orthotopic ID8-Defb29-VEGF tumors. (3) Results: Treatment efficacy was observed both in vitro and in vivo. (4) Conclusions: Our data highlight the need for further investigations to assess the potential of using NIR-PIT for ovarian cancer therapy to improve the treatment outcome of ovarian cancer.

Exploiting the tumor microenvironment for theranostic imaging.

The integration of chemistry and molecular biology with imaging is providing some of the most exciting opportunities in the treatment of cancer. The field of theranostic imaging, where diagnosis is combined with therapy, is particularly suitable for a disease as complex as cancer, especially now that genomic and proteomic profiling can provide an extensive 'fingerprint' of each tumor. Using this information, theranostic agents can be shaped for personalized treatment to target specific compartments, such as the tumor microenvironment (TME), whilst minimizing damage to normal tissue. These theranostic agents can also be used to target multiple pathways or networks by incorporating multiple small interfering RNAs (siRNAs) within a single agent. A decade ago genetic alterations were the primary focus in cancer research. Now it is apparent that the tumor physiological microenvironment, interactions between cancer cells and stromal cells, such as endothelial cells, fibroblasts and macrophages, the extracellular matrix (ECM), and a host of secreted factors and cytokines, influence progression to metastatic disease, aggressiveness and the response of the disease to treatment. In this review, we outline some of the characteristics of the TME, describe the theranostic agents currently available to target the TME and discuss the unique opportunities the TME provides for the design of novel theranostic agents for cancer therapy.

MRI of metastasis-permissive microenvironments.

One of the earliest documented observations of the importance of the microenvironment in metastasis was made by Stephen Paget in 1889. More than a century later, the metastatic cascade remains a major cause of mortality from cancer. Cancer meets the criterion of a successful organization that is able to survive by adapting to changing environments. In fact, the tumor microenvironment and stroma are co-opted and shaped by cancer cells to derive a survival advantage. Cohesive strategies integrating advances in molecular biology and chemistry, with noninvasive multimodality imaging, provide new insights into the role of the tumor microenvironment in promoting metastasis from primary tumors as well as insights into environments that attract and permit cancer cells to establish colonies in distant organs. This article provides an overview of molecular and functional imaging characterization of microenvironments that can promote or permit cancer cells to metastasize and the microenvironmental characteristics of distant metastases.

Protection against cerebral malaria by the low-molecular-weight thiol pantethine.

We report that administration of the low-molecular-weight thiol pantethine prevented the cerebral syndrome in Plasmodium berghei ANKA-infected mice. The protection was associated with an impairment of the host response to the infection, with in particular a decrease of circulating microparticles and preservation of the blood-brain barrier integrity. Parasite development was unaffected. Pantethine modulated one of the early steps of the inflammation-coagulation cascade, i.e., the transbilayer translocation of phosphatidylserine at the cell surface that we demonstrated on red blood cells and platelets. In this, pantethine mimicked the inactivation of the ATP-binding-cassette transporter A1 (ABCA1), which also prevents the cerebral syndrome in this malaria model. However, pantethine acts through a different pathway, because ABCA1 activity was unaffected by the treatment. The mechanisms of pantethine action were investigated, using the intact molecule and its constituents. The disulfide group (oxidized form) is necessary to lower the platelet response to activation by thrombin and collagen. Thio-sensitive mechanisms are also involved in the impairment of microparticle release by TNF-activated endothelial cells. In isolated cells, the effects were obtained by cystamine that lacks the pantothenic moiety of the molecule; however, the complete molecule is necessary to protect against cerebral malaria. Pantethine is well tolerated, and it has already been administered in other contexts to man with limited side effects. Therefore, trials of pantethine treatment in adjunctive therapy for severe malaria are warranted.

The PD-L1 metabolic interactome intersects with choline metabolism and inflammation.

BACKGROUND: Harnessing the power of the immune system by using immune checkpoint inhibitors has resulted in some of the most exciting advances in cancer treatment. The full potential of this approach has, however, not been fully realized for treating many cancers such as pancreatic and breast cancer. Cancer metabolism influences many aspects of cancer progression including immune surveillance. An expanded understanding of how cancer metabolism can directly impact immune checkpoints may allow further optimization of immunotherapy. We therefore investigated, for the first time, the relationship between the overexpression of choline kinase-α (Chk-α), an enzyme observed in most cancers, and the expression of the immune checkpoint PD-L1. METHODS: We used small interfering RNA to downregulate Chk-α, PD-L1, or both in two triple-negative human breast cancer cell lines (MDA-MB-231 and SUM-149) and two human pancreatic ductal adenocarcinoma cell lines (Pa09C and Pa20C). The effects of the downregulation were studied at the genomic, proteomic, and metabolomic levels. The findings were compared with the results obtained by the analysis of public data from The Cancer Genome Atlas Program. RESULTS: We identified an inverse dependence between Chk-α and PD-L1 at the genomic, proteomic, and metabolomic levels. We also found that prostaglandin-endoperoxide synthase 2 (COX-2) and transforming growth factor beta (TGF-ß) play an important role in this relationship. We independently confirmed this relationship in human cancers by analyzing data from The Cancer Genome Atlas Program. CONCLUSIONS: Our data identified previously unknown roles of PD-L1 in cancer cell metabolic reprogramming, and revealed the immunosuppressive increased PD-L1 effect of Chk-α downregulation. These data suggest that PD-L1 regulation of metabolism may be mediated through Chk-α, COX-2, and TGF-ß. The observations provide new insights that can be applied to the rational design of combinatorial therapies targeting immune checkpoints and cancer metabolism.

Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells.

A direct correlation exists between increased choline kinase (Chk) expression, and the resulting increase of phosphocholine levels, and histological tumor grade. To better understand the function of Chk and choline phospholipid metabolism in breast cancer we have stably overexpressed one of the two isoforms of Chk-alpha known to be upregulated in malignant cells, in non-invasive MCF-7 human breast cancer cells. Dynamic tracking of cell invasion and cell metabolism were studied with a magnetic resonance (MR) compatible cell perfusion assay. The MR based invasion assay demonstrated that MCF-7 cells overexpressing Chk-alpha (MCF-7-Chk) exhibited an increase of invasion relative to control MCF-7 cells (0.84 vs 0.3). Proton MR spectroscopy studies showed significantly higher phosphocholine and elevated triglyceride signals in Chk overexpressing clones compared to control cells. A test of drug resistance in MCF-7-Chk cells revealed that these cells had an increased resistance to 5-fluorouracil and higher expression of thymidylate synthase compared to control MCF-7 cells. To further characterize increased drug resistance in these cells, we performed rhodamine-123 efflux studies to evaluate drug efflux pumps. MCF-7-Chk cells effluxed twice as much rhodamine-123 compared to MCF-7 cells. Chk-alpha overexpression resulted in MCF-7 human breast cancer cells acquiring an increasingly aggressive phenotype, supporting the role of Chk-alpha in mediating invasion and drug resistance, and the use of phosphocholine as a biomarker of aggressive breast cancers.

Water and Collagen Content Are High in Pancreatic Cancer: Implications for Quantitative Metabolic Imaging.

In magnetic resonance metabolic imaging, signal from the water content is frequently used for normalization to derive quantitative or semi-quantitative values of metabolites in vivo or ex vivo tumors and tissues. Ex vivo high-resolution metabolic characterization of tumors with magnetic resonance spectroscopy (MRS) provides valuable information that can be used to drive the development of noninvasive MRS biomarkers and to identify metabolic therapeutic targets. Variability in the water content between tumor and normal tissue can result in over or underestimation of metabolite concentrations when assuming a constant water content. Assuming a constant water content can lead to masking of differences between malignant and normal tissues both in vivo and ex vivo. There is a critical need to develop biomarkers to detect pancreatic cancer and to develop novel treatments. Our purpose here was to determine the differences in water content between pancreatic tumors and normal pancreatic tissue as well as other organs to accurately quantify metabolic differences when using the water signal for normalization. Our data identify the importance of factoring the differences in water content between tumors and organs. High-resolution proton spectra of tumors and pancreatic tissue extracts normalized to the water signal, assuming similar water content, did not reflect the significantly increased total choline observed in tumors in vivo without factoring the differences in water content. We identified significant differences in the collagen 1 content between Panc1 and BxPC3 pancreatic tumors and the pancreas that can contribute to the differences in water content that were observed.

Theranostic small interfering RNA nanoparticles in cancer precision nanomedicine.

Due to their ability to effectively downregulate the expression of target genes, small interfering RNA (siRNA) have emerged as promising candidates for precision medicine in cancer. Although some siRNA-based treatments have advanced to clinical trials, challenges such as poor stability during circulation, and less than optimal pharmacokinetics and biodistribution of siRNA in vivo present barriers to the systemic delivery of siRNA. In recent years, theranostic nanomedicine integrating siRNA delivery has attracted significant attention for precision medicine. Theranostic nanomedicine takes advantage of the high capacity of nanoplatforms to ferry cargo with imaging and therapeutic capabilities. These theranostic nanoplatforms have the potential to play a major role in gene specific treatments. Here we have reviewed recent advances in the use of theranostic nanoplatforms to deliver siRNA, and discussed the opportunities as well as challenges associated with this exciting technology. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Implantable Materials and Surgical Technologies > Nanomaterials and Implants.

PD-L1 siRNA Theranostics With a Dextran Nanoparticle Highlights the Importance of Nanoparticle Delivery for Effective Tumor PD-L1 Downregulation.

PURPOSE: The inhibition of immune checkpoints such as programmed cell death ligand-1 (PD-L1/CD274) with antibodies is providing novel opportunities to expose cancer cells to the immune system. Antibody based checkpoint blockade can, however, result in serious autoimmune complications because normal tissues also express immune checkpoints. As sequence-specific gene-silencing agents, the availability of siRNA has significantly expanded the specificity and range of "druggable" targets making them promising agents for precision medicine in cancer. Here, we have demonstrated the ability of a novel biodegradable dextran based theranostic nanoparticle (NP) to deliver siRNA downregulating PD-L1 in tumors. Optical imaging highlighted the importance of NP delivery and accumulation in tumors to achieve effective downregulation with siRNA NPs, and demonstrated low delivery and accumulation in several PD-L1 expressing normal tissues. METHODS: The dextran scaffold was functionalized with small molecules containing amine groups through acetal bonds. The NP was decorated with a Cy5.5 NIR probe allowing visualization of NP delivery, accumulation, and biodistribution. MDA-MB-231 triple negative human breast cancer cells were inoculated orthotopically or subcutaneously to achieve differences in vascular delivery in the tumors. Molecular characterization of PD-L1 mRNA and protein expression in cancer cells and tumors was performed with qRT-PCR and immunoblot analysis. RESULTS: The PD-L1 siRNA dextran NPs effectively downregulated PD-L1 in MDA-MB-231 cells. We identified a significant correlation between NP delivery and accumulation, and the extent of PD-L1 downregulation, with in vivo imaging. The size of the NP of ~ 20 nm allowed delivery through leaky tumor vasculature but not through the vasculature of high PD-L1 expressing normal tissue such as the spleen and lungs. CONCLUSIONS: Here we have demonstrated, for the first time, the feasibility of downregulating PD-L1 in tumors using siRNA delivered with a biodegradable dextran polymer that was decorated with an imaging reporter. Our data demonstrate the importance of tumor NP delivery and accumulation in achieving effective downregulation, highlighting the importance of imaging in siRNA NP delivery. Effective delivery of these siRNA carrying NPs in the tumor but not in normal tissues may mitigate some of the side-effects of immune checkpoint inhibitors by sparing PD-L1 inhibition in these tissues.

Brain metabolites in cholinergic and glutamatergic pathways are altered by pancreatic cancer cachexia.

BACKGROUND: Cachexia is a major cause of morbidity in pancreatic ductal adenocarcinoma (PDAC) patients. Our purpose was to understand the impact of PDAC-induced cachexia on brain metabolism in PDAC xenograft studies, to gain new insights into the causes of cachexia-induced morbidity. Changes in mouse and human plasma metabolites were characterized to identify underlying causes of brain metabolic changes. METHODS: We quantified metabolites, detected with high-resolution 1 H magnetic resonance spectroscopy, in the brain and plasma of normal mice (n = 10) and mice bearing cachexia (n = 10) or non-cachexia (n = 9) inducing PDAC xenografts as well as in human plasma obtained from normal individuals (n = 24) and from individuals with benign pancreatic disease (n = 20) and PDAC (n = 20). Statistical significance was defined as a P value ≤0.05. RESULTS: The brain metabolic signature of cachexia-inducing PDAC was characterized by a significant depletion of choline of -27% and -21% as well as increases of glutamine of 13% and 9% and formate of 21% and 14%, relative to normal controls and non-cachectic tumour-bearing mice, respectively. Good to moderate correlations with percent weight change were found for choline (r = 0.70), glutamine (r = -0.58), and formate (r = -0.43). Significant choline depletion of -38% and -30%, relative to normal controls and non-cachectic tumour-bearing mice, respectively, detected in the plasma of cachectic mice likely contributed to decreased brain choline in cachectic mice. Similarly, relative to normal controls and patients with benign disease, choline levels in human plasma samples of PDAC patients were significantly lower by -12% and -20% respectively. A comparison of plasma metabolites from PDAC patients with and without weight loss identified significant changes in glutamine metabolism. CONCLUSIONS: Disturbances in metabolites of the choline/cholinergic and glutamine/glutamate/glutamatergic neurotransmitter pathways may contribute to morbidity. Metabolic normalization may provide strategies to reduce morbidity. The human plasma metabolite changes observed may lead to the development of companion diagnostic markers to detect PDAC and PDAC-induced cachexia.