Clinically Translatable Hyperpolarized 13C Bicarbonate pH Imaging Method for Use in Prostate Cancer.

Solid tumors such as prostate cancer (PCa) commonly develop an acidic microenvironment with pH 6.5-7.2, owing to heterogeneous perfusion, high metabolic activity, and rapid cell proliferation. In preclinical prostate cancer models, disease progression is associated with a decrease in tumor extracellular pH, suggesting that pH imaging may reflect an imaging biomarker to detect aggressive and high-risk disease. Therefore, we developed a hyperpolarized carbon-13 MRI method to image the tumor extracellular pH (pHe) and prepared it for clinical translation for detection and risk stratification of PCa. This method relies on the rapid breakdown of hyperpolarized (HP) 1,2-glycerol carbonate (carbonyl-13C) via base-catalyzed hydrolysis to produce HP 13CO32-, which is neutralized and converted to HP H13CO3-. After injection, HP H13CO3- equilibrates with HP 13CO2 in vivo and enables the imaging of pHe. Using insights gleaned from mechanistic studies performed in the hyperpolarized state, we solved issues of polarization loss during preparation in a clinical polarizer system. We successfully customized a reaction apparatus suitable for clinical application, developed clinical standard operating procedures, and validated the radiofrequency pulse sequence and imaging data acquisition with a wide range of animal models. The results demonstrated that we can routinely produce a highly polarized and safe HP H13CO3- contrast agent suitable for human injection. Preclinical imaging studies validated the reliability and accuracy of measuring acidification in healthy kidney and prostate tumor tissue. These methods were used to support an Investigational New Drug application to the U.S. Food and Drug Administration. This methodology is now ready to be implemented in human trials, with the ultimate goal of improving the management of PCa.

A machine learning approach that measures pH using acidoCEST MRI of iopamidol.

Tumor acidosis is an important biomarker for aggressive tumors, and extracellular pH (pHe) of the tumor microenvironment can be used to predict and evaluate tumor responses to chemotherapy and immunotherapy. AcidoCEST MRI measures tumor pHe by exploiting the pH-dependent chemical exchange saturation transfer (CEST) effect of iopamidol, an exogenous CT agent repurposed for CEST MRI. However, all pH fitting methodologies for acidoCEST MRI data have limitations. Herein we present results of the application of machine learning for extracting pH values from CEST Z-spectra of iopamidol. We acquired 36,000 experimental CEST spectra from 200 phantoms of iopamidol prepared at five concentrations, five T1 values, and eight pH values at five temperatures, acquired at six saturation powers and six saturation times. We also acquired T1 , T2 , B1 RF power, and B0 magnetic field strength supplementary MR information. These MR images were used to train and validate machine learning models for the tasks of pH classification and pH regression. Specifically, we tested the L1-penalized logistic regression classification (LRC) model and the random forest classification (RFC) model for classifying the CEST Z-spectra for thresholds at pH 6.5 and 7.0. Our results showed that both RFC and LRC were effective for pH classification, although the RFC model achieved higher predictive value, and improved the accuracy of classification accuracy with CEST Z-spectra with a more limited set of saturation frequencies. Furthermore, we used LASSO and random forest regression (RFR) models to explore pH regression, which showed that the RFR model achieved higher accuracy and precision for estimating pH across the entire pH range of 6.2-7.3, especially when using a more limited set of features. Based on these results, machine learning for analysis of acidoCEST MRI is promising for eventual in vivo determination of tumor pHe.

Application of tumor pH/hypoxia-responsive nanoparticles for combined photodynamic therapy and hypoxia-activated chemotherapy.

Introduction: Cancer selectivity, including targeted internalization and accelerated drug release in tumor cells, remains a major challenge for designing novel stimuli-responsive nanocarriers to promote therapeutic efficacy. The hypoxic microenvironment created by photodynamic therapy (PDT) is believed to play a critical role in chemoresistance. Methods: We construct dual-responsive carriers (DANPCT) that encapsulate the photosensitizer chlorin e6 (Ce6) and hypoxia-activated prodrug tirapazamine (TPZ) to enable efficient PDT and PDT-boosted hypoxia-activated chemotherapy. Results and discussion: Due to TAT masking, DANPCT prolonged payload circulation in the bloodstream, and selective tumor cell uptake occurred via acidity-triggered TAT presentation. PDT was performed with a spatially controlled 660-nm laser to enable precise cell killing and exacerbate hypoxia. Hypoxia-responsive conversion of the hydrophobic NI moiety led to the disassembly of DANPCT, facilitating TPZ release. TPZ was reduced to cytotoxic radicals under hypoxic conditions, contributing to the chemotherapeutic cascade. This work offers a sophisticated strategy for programmed chemo-PDT.

Delivery of Chemotherapy Agents and Nucleic Acids with pH-Dependent Nanoparticles.

With less than one percent of systemically injected nanoparticles accumulating in tumors, several novel approaches have been spurred to direct and release the therapy in or near tumors. One such approach depends on the acidic pH of the extracellular matrix and endosomes of the tumor. With an average pH of 6.8, the extracellular tumor matrix provides a gradient for pH-responsive particles to accumulate, enabling greater specificity. Upon uptake by tumor cells, nanoparticles are further exposed to lower pHs, reaching a pH of 5 in late endosomes. Based on these two acidic environments in the tumor, various pH-dependent targeting strategies have been employed to release chemotherapy or the combination of chemotherapy and nucleic acids from macromolecules such as the keratin protein or polymeric nanoparticles. We will review these release strategies, including pH-sensitive linkages between the carrier and hydrophobic chemotherapy agent, the protonation and disruption of polymeric nanoparticles, an amalgam of these first two approaches, and the release of polymers shielding drug-loaded nanoparticles. While several pH-sensitive strategies have demonstrated marked antitumor efficacy in preclinical trials, many studies are early in their development with several obstacles that may limit their clinical use.

Eradication of tumors and development of anti-cancer immunity using STINGa targeted by pHLIP.

Despite significant progress in the development of novel STING agonists (STINGa), applications appear to be challenged by the low efficiency and poor selectivity of these agents. A pH Low Insertion Peptide (pHLIP) extends the lifetime of a STINGa in the blood and targets it to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs) and dendritic cells (DCs). CAFs constitute 25% of all live cells within CT26 tumors, and M2-type TAMs and mMDSCs are the most abundant among the immune cells. The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed (the number of CAFs was reduced by 98%), intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. Further, no tumors developed in 20 out of 25 tumor-free mice re-challenged by an additional injection of cancer cells. The therapeutic effect on CT26 tumors was insignificant in nude mice, lacking T-cells. Thus, targeted delivery of STINGa to tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T-cells, which gain access to the tumor core. The cytotoxic activity of T-cells is not impaired by an acidic environment and immune memory is developed.

pH-Sensitive Targeting of Tumors with Chemotherapy-Laden Nanoparticles: Progress and Challenges.

Accumulating chemotherapeutic drugs such as doxorubicin within a tumor while limiting the drug dose to normal tissues is a central goal of drug delivery with nanoparticles. Liposomal products such as Doxil® represent one of the marked successes of nanoparticle-based strategies. To replicate this success for cancer treatment, many approaches with nanoparticles are being explored in order to direct and release chemotherapeutic agents to achieve higher accumulation in tumors. A promising approach has been stimulus-based therapy, such as the release of chemotherapeutic agents from the nanoparticles in the acidic environments of the tumor matrix or the tumor endosomes. Upon reaching the acidic environments of the tumor, the particles, which are made up of pH-dependent polymers, become charged and release the entrapped chemotherapy agents. This review discusses recent advances in and prospects for pH-dependent histidine-based nanoparticles that deliver chemotherapeutic agents to tumors. The strategies used by investigators include an array of histidine-containing peptides and polymers which form micelles, mixed micelles, nanovesicles, polyplexes, and coat particles. To date, several promising histidine-based nanoparticles have been demonstrated to produce marked inhibition of tumor growth, but challenges remain for successful outcomes in clinical trials. The lessons learned from these histidine-containing particles will provide insight in the development of improved pH-dependent polymeric delivery systems for chemotherapy.

Colorectal cancer extracellular acidosis decreases immune cell killing and is partially ameliorated by pH-modulating agents that modify tumor cell cytokine profiles.

Tumor cells upregulate myriad proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment (TME). Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how extrinsic alterations in pH modulate the interactions between immune cells and tumors cells will help elucidate opportunities for new therapeutic approaches. We observed that pH impacts the function of immune cells, both natural killer (NK) and T cells, which is relevant in the context of a highly acidic TME. Decreased NK and T cell activity was correlated with decreasing pH in a co-culture immune cell-mediated tumor cell-killing assay. The addition of pH-modulating drugs cariporide, lansoprazole, and acetazolamide to the co-culture assay was able to partially mitigate this dampened immune cell function. Treatment of colorectal cancer (CRC) cells with NHE1 inhibitor cariporide increased CRC cell-secreted cytokines involved in immune cell recruitment and activation and decreased cytokines involved in epithelial-mesenchymal transition (EMT). Cariporide treatment also decreased CRC cell shed TRAIL-R2, TRAIL-R3, and PD-L1 which is relevant in the context of immunotherapy. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response in solid tumors such as colorectal cancer.

A pH-responsive photoacoustic imaging probe for tumor pH imaging in vivo based on polyaniline-bovine serum albumin.

Precise pH detection in tumors can guide the design of pH-responsive drugs and theranostic agents to improve treatment efficacy. However, most reported pH-responsive probes are fluorescent probes, for which in vivo application is limited by low probe penetration depth. In this study, a pH-responsive polyaniline-bovine serum albumin (BSA) probe was constructed for precise pH detection in tumors using photoacoustic imaging. The probe can be used to generate high-resolution images of deep biological tissues. The photoacoustic signal of the polyaniline-BSA probe exhibits a clear linear relationship with pH in the range of 5-6.8 both in vitro and in vivo, indicating that the probe is ideal for precise pH detection in most tumors. The polyaniline-BSA probe also exhibits satisfactory biocompatibility, low toxicity, fast response, and good reversibility. This work provides a useful in vivo pH detection probe for developing pH-responsive drugs and theranostic agents.

Hyperthermia can alter tumor physiology and improve chemo- and radio-therapy efficacy.

Hyperthermia has demonstrated clinical success in improving the efficacy of both chemo- and radio-therapy in solid tumors. Pre-clinical and clinical research studies have demonstrated that targeted hyperthermia can increase tumor blood flow and increase the perfused fraction of the tumor in a temperature and time dependent manner. Changes in tumor blood circulation can produce significant physiological changes including enhanced vascular permeability, increased oxygenation, decreased interstitial fluid pressure, and reestablishment of normal physiological pH conditions. These alterations in tumor physiology can positively impact both small molecule and nanomedicine chemotherapy accumulation and distribution within the tumor, as well as the fraction of the tumor susceptible to radiation therapy. Hyperthermia can trigger drug release from thermosensitive formulations and further improve the accumulation, distribution, and efficacy of chemotherapy.

Stepwise-activatable hypoxia triggered nanocarrier-based photodynamic therapy for effective synergistic bioreductive chemotherapy.

Tumor cell populations are highly heterogeneous, which limit the homogeneous distribution and optimal delivery of nanomedicines, thereby inducing insufficient therapeutic benefits. We develop tumor microenvironment activatable and external stimuli-responsive drug delivery system (TAT+AzoNPs), which can improve photodynamic therapy (PDT) induced bioreductive chemotherapy in different tumor cells both proximal and distal to vessels. The TAT peptide on the surface of TAT+AzoNPs can both facilitate the cell uptake and the penetration of TAT+AzoNPs, owing to its responsiveness to tumor stimuli pH. TAT+AzoNPs can keep the cargoes (photosensitizer chlorine e6 (Ce6) and hypoxia activatable prodrug tirapazamine (TPZ)) and highly accumulate within tumor cells proximity and distal to vessels. The Azo-benzene bonds as the linkers between amphiphilic polymers remain stable under normoxia, but quite break at hypoxic conditions. Upon external laser irradiation, the intratumoral fate of TAT+AzoNPs involved two processes: 1) TAT+AzoNPs achieve efficient PDT on tumor cells proximal to vessel, since sufficient O2 supply; and 2) PDT-induced more hypoxia can trigger TPZ release by breakage of Azo-benzene bond as well as accelerate the activation of TPZ for improvingcombination therapy efficacy in tumor cells distal to vessel. This study gives a direction for the development of stepwise-activatable hypoxia triggered nanosystem for PDT-induced bioreductive chemotherapy for tumor cells in different distances to vessels.

Non-invasive Investigation of Tumor Metabolism and Acidosis by MRI-CEST Imaging.

Altered metabolism is considered a core hallmark of cancer. By monitoring in vivo metabolites changes or characterizing the tumor microenvironment, non-invasive imaging approaches play a fundamental role in elucidating several aspects of tumor biology. Within the magnetic resonance imaging (MRI) modality, the chemical exchange saturation transfer (CEST) approach has emerged as a new technique that provides high spatial resolution and sensitivity for in vivo imaging of tumor metabolism and acidosis. This mini-review describes CEST-based methods to non-invasively investigate tumor metabolism and important metabolites involved, such as glucose and lactate, as well as measurement of tumor acidosis. Approaches that have been exploited to assess response to anticancer therapies will also be reported for each specific technique.

Targeted inhibition of MCT4 disrupts intracellular pH homeostasis and confers self-regulated apoptosis on hepatocellular carcinoma.

The high lactate production rate in hepatocellular carcinoma cells (HCC) have a profound impact on their malignant properties. In adaptation to the enhanced lactate stress, lactate-effusing monocarboxylate transporter 4(MCT4) is usually overexpressed in a broad range of HCC subtypes. In this study, the MCT4-mediated lactate efflux in HCC was blocked using microRNA-145(miR-145), which would force the endogenously generated lactate to accumulate within tumor cells in a self-regulated manner, resulting in the acidification of the cytoplasmic compartment as well as partial neutralization for pH in the tumor extracellular environment. Evaluations on multiple representative HCC subtypes (HepG2, Hep3B and HuH7) suggested that the disrupted pH homeostasis would amplify the lactate stress to initiate HCC apoptosis, while at the same time also suppressing their migration and invasion abilities. Moreover, safety tests on 7702 cells and living animals revealed that MCT4-blockade treatment has no cytotoxicity against healthy cells/tissues. The results indicate the MCT4-inhibition-induced disruption of tumor intracellular pH holds promise as a therapy against not only HCC, but a broader spectrum of MCT4-overexpressing hyperglycolytic tumors.

Tumor pH-Responsive Albumin/Polyaniline Assemblies for Amplified Photoacoustic Imaging and Augmented Photothermal Therapy.

Tumor-microenvironment-responsive theranostics have great potential for precision diagnosis and effective treatment of cancer. Polyaniline (PANI) is the first reported pH-responsive organic photothermal agent and is widely used as a theranostic agent. However, tumor pH-responsive PANI-based theranostic agents are not explored, mainly because the conversion from the emeraldine base (EB) to emeraldine salt (ES) state of PANI requires pH < 4, which is lower than tumor acidic microenvironment. Herein, a tumor pH-responsive PANI-based theranostic agent is designed and prepared for amplified photoacoustic imaging guided augmented photothermal therapy (PTT), through intermolecular acid-base reactions between carboxyl groups of bovine serum albumin (BSA) and imine moieties of PANI. The albumin/PANI assemblies (BSA-PANI) can convert from the EB to ES state at pH < 7, accompanied by the absorbance redshift from visible to near-infrared region. Both in vitro and in vivo results demonstrate that tumor acidic microenvironment can trigger both the photoacoustic imaging (PAI) signal amplification and the PTT efficacy enhancement of BSA-PANI assemblies. This work not only highlights that BSA-PANI assemblies overcome the limitation of low-pH protonation, but also provides a facile assembly strategy for a tumor pH-responsive PANI-based nanoplatform for cancer theranostics.

pH-responsive hyaluronate-anchored extracellular vesicles to promote tumor-targeted drug delivery.

pH-Responsive drug vehicles targeting the specific extracellular pH of tumors have served as potent tools to overcome the limitation (e.g., low tumor seletivity) in antitumor drug delivery system. Here, we describe the advantage of pH-responsive extracellular vesicles (HDEA@EVs) containing the hyaluronic acid grafted with 3-(diethylamino)propylamine (HDEA) and a model antitumor drug, doxorubicin (DOX). We demonstrated their physicochemical characteristics through in vitro cell endocytosis, in vitro tumor cell toxicity, in vivo biodistribution, and in vivo tumor regression efficacy experiments. Because the HDEA@EVs efficiently responded to extracellular tumor pH (pH 6.5) and actively bound to CD44 receptors on HCT-116 tumor cells, the EVs selectively inhibited CD44+ tumor cell growth in vitro, and CD44+ tumor development in vivo. From these results, we conclude that HDEA@EVs can help in designing effective strategies for pharmacologic intervention in tumor therapy.

Programmed Nanoparticle-Loaded Nanoparticles for Deep-Penetrating 3D Cancer Therapy.

Tumors are 3D, composed of cellular agglomerations and blood vessels. Therapies involving nanoparticles utilize specific accumulations due to the leaky vascular structures. However, systemically injected nanoparticles are mostly uptaken by cells located on the surfaces of cancer tissues, lacking deep penetration into the core cancer regions. Herein, an unprecedented strategy, described as injecting "nanoparticle-loaded nanoparticles" to address the long-lasting problem is reported for effective surface-to-core drug delivery in entire 3D tumors. The "nanoparticle-loaded nanoparticle" is a silica nanoparticle (≈150 nm) with well-developed, interconnected channels (diameter of ≈30 nm), in which small gold nanoparticles (AuNPs) (≈15 nm) with programmable DNA are located. The nanoparticle (AuNPs)-loaded nanoparticles (silica): (1) can accumulate in tumors through leaky vascular structures by protecting the inner therapeutic AuNPs during blood circulation, and then (2) allow diffusion of the AuNPs for penetration into the entire surface-to-core tumor tissues, and finally (3) release a drug triggered by cancer-characteristic pH gradients. The hierarchical "nanoparticle-loaded nanoparticle" can be a rational design for cancer therapies because the outer large nanoparticles are effective in blood circulation and in protection of the therapeutic nanoparticles inside, allowing the loaded small nanoparticles to penetrate deeply into 3D tumors with anticancer drugs.

QUESPOWR MRI: QUantification of Exchange as a function of Saturation Power On the Water Resonance.

QUantification of Exchange as a function of Saturation Power On the Water Resonance (QUESPOWR) MRI is a new method that can estimate chemical exchange rates. This method acquires a series of OPARACHEE MRI acquisitions with a range of RF powers for the WALTZ16(∗) pulse train, which are applied on the water resonance. A QUESPOWR plot can be generated from the power dependence of the % water signal, which is similar to a QUESP plot that is generated from CEST MRI acquisition methods with RF saturation applied off-resonance from water. A QUESPOWR plot can be quantitatively analyzed using linear fitting methods to provide estimates of average chemical exchange rates. Analyses of the shapes of QUESPOWR plots can also be used to estimate relative differences in average chemical exchange rates and concentrations of biomolecules. The performance of QUESPOWR MRI was assessed via simulations, an in vitro study with iopamidol, and an in vivo study with a mouse model of mammary carcinoma. The results showed that QUESPOWR MRI is especially sensitive to chemical exchange between water and biomolecules that have intermediate to fast chemical exchange rates and chemical shifts that are close to water, which are notoriously difficult to assess with other CEST MRI methods. In addition, in vivo QUESPOWR MRI detected acidic tumor tissues relative to normal tissues that are pH-neutral, and therefore may be a new paradigm for tumor detection with MRI.

Tumor acidity-sensitive linkage-bridged block copolymer for therapeutic siRNA delivery.

The design of ideal nanoparticle delivery systems should be capable of meeting the requirements of several stages of drug delivery, including prolonged circulation, enhanced accumulation and penetration in the tumor, facilitated cellular internalization and rapid release of the active drug in the tumor cells. However, among the current design strategies, meeting the requirements of one stage often conflicts with the other. Herein, a tumor pH-labile linkage-bridged block copolymer of poly(ethylene glycol) with poly(lacide-co-glycolide) (PEG-Dlinkm-PLGA) was used for siRNA delivery to fulfill all aforementioned requirements of these delivery stages. The obtained siRNA-encapsulating PEG-Dlinkm-PLGA nanoparticle gained efficiently prolonged circulation in the blood and preferential accumulation in tumor sites via the PEGylation. Furthermore, the PEG surface layer was detached in response to the tumor acidic microenvironment to facilitate cellular uptake, and the siRNA was rapidly released within tumor cells due to the hydrophobic PLGA layer. Hence, PEG-Dlinkm-PLGA nanoparticles met the requirements of several stages of drug delivery, and resulted in the enhanced therapeutic effect of the nanoparticular delivery systems.

Metabolic changes associated with tumor metastasis, part 1: tumor pH, glycolysis and the pentose phosphate pathway.

Metabolic adaptations are intimately associated with changes in cell behavior. Cancers are characterized by a high metabolic plasticity resulting from mutations and the selection of metabolic phenotypes conferring growth and invasive advantages. While metabolic plasticity allows cancer cells to cope with various microenvironmental situations that can be encountered in a primary tumor, there is increasing evidence that metabolism is also a major driver of cancer metastasis. Rather than a general switch promoting metastasis as a whole, a succession of metabolic adaptations is more likely needed to promote different steps of the metastatic process. This review addresses the contribution of pH, glycolysis and the pentose phosphate pathway, and a companion paper summarizes current knowledge regarding the contribution of mitochondria, lipids and amino acid metabolism. Extracellular acidification, intracellular alkalinization, the glycolytic enzyme phosphoglucose isomerase acting as an autocrine cytokine, lactate and the pentose phosphate pathway are emerging as important factors controlling cancer metastasis.

Evaluations of extracellular pH within in vivo tumors using acidoCEST MRI.

PURPOSE: A practical, noninvasive method is needed to measure the extracellular pH (pHe) within in vivo tumors to longitudinally monitor tumor acidosis. We have optimized a biomedical imaging method, termed acidoCEST MRI, to provide noninvasive assessments of tumor pHe in preclinical models of mammary carcinoma. METHODS: A CEST-FISP MRI method was optimized to detect the chemical exchange saturation transfer (CEST) of two amide protons of a clinically approved CT contrast agent, iopromide. The ratio of the two CEST effects was used to measure pH. Routes of administration of iopromide were evaluated to ensure sufficient delivery of the agent to the tumor. The optimized acidoCEST MRI method was then used to evaluate the change in tumor pHe following alkalinizing bicarbonate treatment. RESULTS: The acidoCEST MRI protocol measured pH between 6.2 and 7.2 pH units. Greater delivery of iopromide was shown to improve the precision of the measurement of tumor pHe, but the agent did not influence the tumor pHe. AcidoCEST MRI was used to longitudinally monitor the effect of bicarbonate treatment on the pHe of tumors and bladders. CONCLUSION: This study demonstrates that an optimized acidoCEST MRI method is a practical, noninvasive method for assessing changes in tumor acidosis.

Rational design of polyion complex nanoparticles to overcome cisplatin resistance in cancer therapy.

Rationally designed PIC nanoparticles as next-generation delivery system: we have developed a core-shell-corona PIC nanoparticle (⊕) NP/Pt@PPC-DA as a next-generation delivery system. (⊕) NP/Pt@PPC-DA exhibits prolonged circulation and enhanced drug accumulation in tumors. Subsequently, tumor pH leads to the release of (⊕) NP/Pt, which facilitates cellular uptake followed by rapid intracellular cisplatin release. Using this delivery strategy cisplatin-resistant tumor growth in a murine xenograft model has been successfully suppressed.