Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors.

Tumor acidosis is associated with increased invasiveness and drug resistance. Here, we take an unbiased approach to identify vulnerabilities of acid-exposed cancer cells by combining pH-dependent flow cytometry cell sorting from 3D colorectal tumor spheroids and transcriptomic profiling. Besides metabolic rewiring, we identify an increase in tetraploid cell frequency and DNA damage response as consistent hallmarks of acid-exposed cancer cells, supported by the activation of ATM and ATR signaling pathways. We find that regardless of the cell replication error status, both ATM and ATR inhibitors exert preferential growth inhibitory effects on acid-exposed cancer cells. The efficacy of a combination of these drugs with 5-FU is further documented in 3D spheroids as well as in patient-derived colorectal tumor organoids. These data position tumor acidosis as a revelator of the therapeutic potential of DNA repair blockers and as an attractive clinical biomarker to predict the response to a combination with chemotherapy.

Obesity and triple-negative-breast-cancer: Is apelin a new key target?

Epidemiological studies have shown that obese subjects have an increased risk of developing triple-negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high-fat diet in TNBC tumour-bearing mice significantly increased tumour growth. By showing no effect of high-fat diet in obesity-resistant mice, we demonstrated the necessity to develop obesity-related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity-related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.

Exploring the Phototoxicity of Hypoxic Active Iridium(III)-Based Sensitizers in 3D Tumor Spheroids.

Among all molecules developed for anticancer therapies, photodynamic therapeutic agents have a unique profile. Their maximal activity is specifically triggered in tumors by light, and toxicity of even systemically delivered drug is prevented in nonilluminated parts of the body. Photosensitizers exert their therapeutic effect by producing reactive oxygen species via a light-activated reaction with molecular oxygen. Consequently, the lowering of pO2 deep in solid tumors limits their treatment and makes essential the design of oxygen-independent sensitizers. In this perspective, we have recently developed Ir(III)-based molecules able to oxidize biomolecules by type I processes under oxygen-free conditions. We examine here their phototoxicity in relevant biological models. We show that drugs, which are mitochondria-accumulated, induce upon light irradiation a dramatic decrease of the cell viability, even under low oxygen conditions. Finally, assays on 3D tumor spheroids highlight the importance of the light-activation step and the oxygen consumption rate on the drug activity.

PAMAM G4.5-chlorin e6 dendrimeric nanoparticles for enhanced photodynamic effects.

There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic photodynamic therapy. Therefore, we aimed to develop covalent conjugates between the photosensitizer chlorin e6 (Ce6) and PAMAM G4.5 dendrimers. Singlet oxygen generation (SOG) efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. Compared to free Ce6, PAMAM anchored Ce6 displays a much higher photodynamic effect, which is ascribable to better internalization in a tumor cell model. Intracellular fate and internalization pathway of our different compounds were investigated using specific inhibition conditions and confocal fluorescence microscopy. Free Ce6 was shown to enter the cells by a simple diffusion mechanism, while G4.5-Ce6-PEG internalization was dependent on the caveolae pathway, whereas G4.5-Ce6 was subjected to the clathrin-mediated endocytosis pathway. Subcellular localization of PAMAM anchored Ce6, PEGylated or not, was very similar suggesting that the nanoparticles behave similarly in the cells. As a conclusion, we have demonstrated that PEGylated G4.5 PAMAM-Ce6 dendrimers may offer effective biocompatible nanoparticles for improved photodynamic treatment in a preclinical tumor model.

3D evaluation of the extracellular matrix of hypoxic pancreatic islets using light sheet fluorescence microscopy.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O2 21%) or hypoxic (O2 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets' functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.

Optogenetically controlled inflammasome activation demonstrates two phases of cell swelling during pyroptosis.

Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1ß and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.

Peroxidation of n-3 and n-6 polyunsaturated fatty acids in the acidic tumor environment leads to ferroptosis-mediated anticancer effects.

Tumor acidosis promotes disease progression through a stimulation of fatty acid (FA) metabolism in cancer cells. Instead of blocking the use of FAs by acidic cancer cells, we examined whether excess uptake of specific FAs could lead to antitumor effects. We found that n-3 but also remarkably n-6 polyunsaturated FA (PUFA) selectively induced ferroptosis in cancer cells under ambient acidosis. Upon exceeding buffering capacity of triglyceride storage into lipid droplets, n-3 and n-6 PUFA peroxidation led to cytotoxic effects in proportion to the number of double bonds and even more so in the presence of diacylglycerol acyltransferase inhibitors (DGATi). Finally, an n-3 long-chain PUFA-rich diet significantly delayed mouse tumor growth when compared with a monounsaturated FA-rich diet, an effect further accentuated by administration of DGATi or ferroptosis inducers. These data point out dietary PUFA as a selective adjuvant antitumor modality that may efficiently complement pharmacological approaches.

Therapy-induced DNA methylation inactivates MCT1 and renders tumor cells vulnerable to MCT4 inhibition.

Metabolic plasticity in cancer cells makes use of metabolism-targeting agents very challenging. Drug-induced metabolic rewiring may, however, uncover vulnerabilities that can be exploited. We report that resistance to glycolysis inhibitor 3-bromopyruvate (3-BrPA) arises from DNA methylation in treated cancer cells and subsequent silencing of the monocarboxylate transporter MCT1. We observe that, unexpectedly, 3-BrPA-resistant cancer cells mostly rely on glycolysis to sustain their growth, with MCT4 as an essential player to support lactate flux. This shift makes cancer cells particularly suited to adapt to hypoxic conditions and resist OXPHOS inhibitors and anti-proliferative chemotherapy. In contrast, blockade of MCT4 activity in 3-BrPA-exposed cancer cells with diclofenac or genetic knockout, inhibits growth of derived spheroids and tumors in mice. This study supports a potential mode of collateral lethality according to which metabolic adaptation of tumor cells to a first-line therapy makes them more responsive to a second-line treatment.

Inhibition of colorectal cancer-associated fibroblasts by lipid nanocapsules loaded with acriflavine or paclitaxel.

Crosstalk between cancer-associated fibroblasts (CAFs) and colorectal cancer cells promotes tumor growth and contributes to chemoresistance. In this study, we assessed the sensitivity of a primary CAF cell line, CT5.3hTERT, to standard-of-care and alternative cytotoxic treatments. Paclitaxel (PTX) and acriflavine (ACF) were identified as the most promising molecules to inhibit CAF development. To allow the translational use of both drugs, we developed lipid nanocapsule (LNC) formulations for PTX and ACF. Finally, we mixed CAFs and tumor cell lines in a cocultured spheroid, and the effect of both drugs was investigated by histological analyses. We demonstrated CAF inhibition by LNC-ACF and whole tumor inhibition by LNC-PTX. Altogether, we proposed a new strategy to reduce CAF populations in the colorectal microenvironment that should be tested in vivo.

TGFß2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells.

Acidosis, a common characteristic of the tumor microenvironment, is associated with alterations in metabolic preferences of cancer cells and progression of the disease. Here we identify the TGF-ß2 isoform at the interface between these observations. We document that acidic pH promotes autocrine TGF-ß2 signaling, which in turn favors the formation of lipid droplets (LD) that represent energy stores readily available to support anoikis resistance and cancer cell invasiveness. We find that, in cancer cells of various origins, acidosis-induced TGF-ß2 activation promotes both partial epithelial-to-mesenchymal transition (EMT) and fatty acid metabolism, the latter supporting Smad2 acetylation. We show that upon TGF-ß2 stimulation, PKC-zeta-mediated translocation of CD36 facilitates the uptake of fatty acids that are either stored as triglycerides in LD through DGAT1 or oxidized to generate ATP to fulfill immediate cellular needs. We also address how, by preventing fatty acid mobilization from LD, distant metastatic spreading may be inhibited.

Preclinical Evaluation of White Led-Activated Non-porphyrinic Photosensitizer OR141 in 3D Tumor Spheroids and Mouse Skin Lesions.

Photodynamic therapy (PDT) is used to treat malignancies and precancerous lesions. Near-infrared light delivered by lasers was thought for a while to be the most appropriate option to activate photosensitizers, mostly porphyrins, in the depth of the diseased tissues. More recently, however, several advantages including low cost and reduced adverse effects led to consider light emitting diodes (LED) and even daylight as an alternative to use PDT to treat accessible lesions. In this study we examined the capacity of OR141, a recently identified non-porphyrin photosensitizer (PS), to exert significant cytotoxic effects in various models of skin lesions and tumors upon white light activation. Using different cancer cell lines, we first identified LED lamp as a particularly suited source of light to maximize anti-proliferative effects of OR141. We then documented that OR141 diffusion and light penetration into tumor spheroids both reached thresholds compatible with the induction of cell death deep inside these 3D culture models. We further identified Arlasove as a clinically suitable solvent for OR141 that we documented by using Franz cells to support significant absorption of the PS through human skin. Finally, using topical but also systemic administration, we validated growth inhibitory effects of LED-activated OR141 in mouse skin tumor xenograft and precancerous lesions models. Altogether these results open clinical perspectives for the use of OR141 as an attractive PS to treat superficial skin malignant and non-malignant lesions using affordable LED lamp for photoactivation.

Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects.

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition.

Antidiabetic Biguanides Radiosensitize Hypoxic Colorectal Cancer Cells Through a Decrease in Oxygen Consumption.

Background and Purpose: The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. However, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. This study therefore examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption. Materials and Methods: A panel of colorectal cancer cells (HCT116, DLD-1, HT29, SW480, and CT26) was exposed to metformin or phenformin for 16 h at indicated concentrations. Afterward, cell viability was measured by MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) was examined by western blot. Mitochondria complexes activity and oxygen consumption rate (OCR) were measured by seahorse analyzer. The radiosensitivity of tumor cells was assessed by colony formation assay under aerobic and hypoxic conditions. The in vitro findings were further validated in colorectal CT26 tumor model. Results: Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced OCR in a dose-dependent manner starting at 3 mM and 30 µM, respectively. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 µM, respectively. Regarding intrinsic radioresistance, both of them did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. In tumor-bearing mice, metformin or phenformin alone did not show any anti-tumor effect. While in combination with radiation, both of them substantially delayed tumor growth and enhanced radioresponse, respectively, by 1.3 and 1.5-fold. Conclusion: Our results demonstrate that metformin and phenformin overcome hypoxic radioresistance through inhibition of mitochondrial respiration, and provide a rationale to explore metformin and phenformin as hypoxic radiosensitizers.

Prospects of in vivo singlet oxygen luminescence monitoring: Kinetics at different locations on living mice.

BACKGROUND: Singlet oxygen observation is considered a valuable tool to assess and optimize PDT treatment. In complex systems, such as tumors in vivo, only the direct, time-resolved singlet oxygen luminescence detection can give reliable information about generation and interaction of singlet oxygen. Up to now, evaluation of kinetics was not possible due to insufficient signal-to-noise ratio. Here we present high signal-to-noise ratio singlet oxygen luminescence kinetics obtained in mouse tumor model under PDT relevant conditions. METHODS: A highly optimized system based on a custom made laser diode excitation source and a high aperture multi-furcated fiber, utilizing a photomultiplier tube with a multi photon counting device was used. RESULTS: Luminescence kinetics with unsurpassed signal-to-noise ratio were gained from tumor bearing nude mice in vivo upon topic application, subcutaneous injection as well as intravenous injection of different photosensitizers (chlorin e6 and dendrimer formulations of chlorin e6). Singlet oxygen kinetics in appropriate model systems are discussed to facilitate the interpretation of complex kinetics obtained from in vivo tumor tissue. CONCLUSIONS: This is the first study addressing the complexity of singlet oxygen luminescence kinetics in tumor tissue. At present, further investigations are needed to fully explain the processes involved. Nevertheless, the high signal-to-noise ratio proves the applicability of direct time-resolved singlet oxygen luminescence detection as a prospective tool for monitoring photodynamic therapy.

Inclusion complexation with ß-cyclodextrin derivatives alters photodynamic activity and biodistribution of meta-tetra(hydroxyphenyl)chlorin.

Application of meta-tetra(hydroxyphenyl)chorin (mTHPC) one of the most effective photosensitizer (PS) in photodynamic therapy of solid tumors encounters several complications resulting from its insolubility in aqueous medium. To improve its solubility and pharmacokinetic properties, two modified ß-cyclodextrins (ß-CDs) methyl-ß-cyclodextrin (M-ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (Hp-ß-CD) were proposed. The aim of this work was to evaluate the effect of ß-CDs on mTHPC behavior at various stages of its distribution in vitro and in vivo. For this purpose, we have studied the influence of the ß-CDs on mTHPC binding to the serum proteins, its accumulation, distribution and photodynamic efficiency in HT29 cells. In addition, the processes of mTHPC biodistribution in HT29 tumor bearing mice after intravenous injection of PS alone or with the ß-CDs were compared. Interaction of mTHPC with studied ß-CDs leads to the formation of inclusion complexes that completely abolishes its aggregation after introduction into serum. It was demonstrated that the ß-CDs have a concentration-dependent effect on the process of mTHPC distribution in blood serum. At high concentrations, ß-CDs can form inclusion complexes with mTHPC in the blood that can have a significant impact on PS distribution out of the vascular system in solid tissues. Besides, the ß-CDs increase diffusion movement of mTHPC molecules that can significantly accelerate the delivery of PS to the targets cells and tissues. In vivo study confirms the fact that the use of ß-CDs allows to modify mTHPC distribution processes in tumor bearing animals that is reflected in the decreased level of PS accumulation in skin and muscles, as well as in the increased PS accumulation in tumor. Further studies are underway to verify the optimal protocols of mTHPC/ß-CD formulation for photodynamic therapy.