Metabolic Profiling to Assess Response to Targeted and Immune Therapy in Melanoma.

There is currently no consensus to determine which advanced melanoma patients will benefit from targeted therapy, immunotherapy, or a combination of both, highlighting the critical need to identify early-response biomarkers to advanced melanoma therapy. The goal of this review is to provide scientific rationale to highlight the potential role of metabolic imaging to assess response to targeted and/or immune therapy in melanoma cancer. For that purpose, a brief overview of current melanoma treatments is provided. Then, current knowledge with respect to melanoma metabolism is described with an emphasis on major crosstalks between melanoma cell metabolism and signaling pathways involved in BRAF-targeted therapy as well as in immune checkpoint inhibition therapies. Finally, preclinical and clinical studies using metabolic imaging and/or profiling to assess response to melanoma treatment are summarized with a particular focus on PET (Positron Emission Tomography) imaging and 13C-MRS (Magnetic Resonance Spectroscopy) methods.

Hyperpolarized 13C-Pyruvate to Assess Response to Anti-PD1 Immune Checkpoint Inhibition in YUMMER 1.7 Melanoma Xenografts.

There is currently no consensus to determine which advanced melanoma patients will benefit from immunotherapy, highlighting the critical need to identify early-response biomarkers to immune checkpoint inhibitors. The aim of this work was to evaluate in vivo metabolic spectroscopy using hyperpolarized (HP) 13C-pyruvate and 13C-glucose to assess early response to anti-PD1 therapy in the YUMMER1.7 syngeneic melanoma model. The xenografts showed a significant tumor growth delay when treated with two cycles of an anti-PD1 antibody compared to an isotype control antibody. 13C-MRS was performed in vivo after the injection of hyperpolarized 13C-pyruvate, at baseline and after one cycle of immunotherapy, to evaluate early dynamic changes in 13C-pyruvate-13C-lactate exchange. Furthermore, ex vivo 13C-MRS metabolic tracing experiments were performed after U-13C-glucose injection following one cycle of immunotherapy. A significant decrease in the ratio of HP 13C-lactate to 13C-pyruvate was observed in vivo in comparison with the isotype control group, while there was a lack of change in the levels of 13C lactate and 13C alanine issued from 13C glucose infusion, following ex vivo assessment on resected tumors. Thus, these results suggest that hyperpolarized 13C-pyruvate could be used to assess early response to immune checkpoint inhibitors in melanoma patients.

EPR Investigations to Study the Impact of Mito-Metformin on the Mitochondrial Function of Prostate Cancer Cells.

BACKGROUND: Mito-metformin10 (MM10), synthesized by attaching a triphenylphosphonium cationic moiety via a 10-carbon aliphatic side chain to metformin, is a mitochondria-targeted analog of metformin that was recently demonstrated to alter mitochondrial function and proliferation in pancreatic ductal adenocarcinoma. Here, we hypothesized that this compound may decrease the oxygen consumption rate (OCR) in prostate cancer cells, increase the level of mitochondrial ROS, alleviate tumor hypoxia, and radiosensitize tumors. METHODS: OCR and mitochondrial superoxide production were assessed by EPR (9 GHz) in vitro in PC-3 and DU-145 prostate cancer cells. Reduced and oxidized glutathione were assessed before and after MM10 exposure. Tumor oxygenation was measured in vivo using 1 GHz EPR oximetry in PC-3 tumor model. Tumors were irradiated at the time of maximal reoxygenation. RESULTS: 24-hours exposure to MM10 significantly decreased the OCR of PC-3 and DU-145 cancer cells. An increase in mitochondrial superoxide levels was observed in PC-3 but not in DU-145 cancer cells, an observation consistent with the differences observed in glutathione levels in both cancer cell lines. In vivo, the tumor oxygenation significantly increased in the PC-3 model (daily injection of 2 mg/kg MM10) 48 and 72 h after initiation of the treatment. Despite the significant effect on tumor hypoxia, MM10 combined to irradiation did not increase the tumor growth delay compared to the irradiation alone. CONCLUSIONS: MM10 altered the OCR in prostate cancer cells. The effect of MM10 on the superoxide level was dependent on the antioxidant capacity of cell line. In vivo, MM10 alleviated tumor hypoxia, yet without consequence in terms of response to irradiation.

Metabolic imaging using hyperpolarized 13 C-pyruvate to assess sensitivity to the B-Raf inhibitor vemurafenib in melanoma cells and xenografts.

Nearly all melanoma patients with a BRAF-activating mutation will develop resistance after an initial clinical benefit from BRAF inhibition (BRAFi). The aim of this work is to evaluate whether metabolic imaging using hyperpolarized (HP) 13 C pyruvate can serve as a metabolic marker of early response to BRAFi in melanoma, by exploiting the metabolic effects of BRAFi. Mice bearing human melanoma xenografts were treated with the BRAFi vemurafenib or vehicle. In vivo HP 13 C magnetic resonance spectroscopy was performed at baseline and 24 hours after treatment to evaluate changes in pyruvate-to-lactate conversion. Oxygen partial pressure was measured via electron paramagnetic resonance oximetry. Ex vivo qRT-PCR, immunohistochemistry and WB analysis were performed on tumour samples collected at the same time-points selected for in vivo experiments. Similar approaches were applied to evaluate the effect of BRAFi on sensitive and resistant melanoma cells in vitro, excluding the role of tumour microenvironment. BRAF inhibition induced a significant increase in the HP pyruvate-to-lactate conversion in vivo, followed by a reduction of hypoxia. Conversely, the conversion was inhibited in vitro, which was consistent with BRAFi-mediated impairment of glycolysis. The paradoxical increase of pyruvate-to-lactate conversion in vivo suggests that such conversion is highly influenced by the tumour microenvironment.

Acetate: Friend or foe against breast tumour growth in the context of obesity?

Acetate is reported as a regulator of fat mass but also as lipogenic source for cancer cells. Breast cancer is surrounded by adipose tissue and has been associated with obesity. However, whether acetate contributes to cancer cell metabolism as lipogenic substrate and/or by changing fat storage and eventually obesity-induced breast cancer progression remains unknown. Therefore, we studied the contribution of acetate to breast cancer metabolism and progression. In vitro, we found that acetate is not a bioenergetic substrate under normoxia and did not result in a significant change of growth. However, by using lipidomic approaches, we discovered that acetate changes the lipid profiles of the cells under hypoxia. Moreover, while mice fed a high-fat diet (HFD) developed bigger tumours than their lean counterparts, exogenous acetate supplementation leads to a complete abolishment of fat mass gain without reverting the HFD-induced obesity-driven tumour progression. In conclusion, although acetate protects against diet-induced obesity, our data suggest that it is not affecting HFD-driven tumour progression.

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.

Non-invasive in vivo imaging of early metabolic tumor response to therapies targeting choline metabolism.

The cholinic phenotype, characterized by elevated phosphocholine and a high production of total-choline (tCho)-containing metabolites, is a metabolic hallmark of cancer. It can be exploited for targeted therapy. Non-invasive imaging biomarkers are required to evaluate an individual's response to targeted anticancer agents that usually do not rapidly cause tumor shrinkage. Because metabolic changes can manifest at earlier stages of therapy than changes in tumor size, the aim of the current study was to evaluate (1)H-MRS and diffusion-weighted MRI (DW-MRI) as markers of tumor response to the modulation of the choline pathway in mammary tumor xenografts. Inhibition of choline kinase activity was achieved with the direct pharmacological inhibitor H-89, indirect inhibitor sorafenib and down-regulation of choline-kinase α (ChKA) expression using specific short-hairpin RNA (shRNA). While all three strategies significantly decreased tCho tumor content in vivo, only sorafenib and anti-ChKA shRNA significantly repressed tumor growth. The increase of apparent-diffusion-coefficient of water (ADCw) measured by DW-MRI, was predictive of the induced necrosis and inhibition of the tumor growth in sorafenib treated mice, while the absence of change in ADC values in H89 treated mice predicted the absence of effect in terms of tumor necrosis and tumor growth. In conclusion, (1)H-choline spectroscopy can be useful as a pharmacodynamic biomarker for choline targeted agents, while DW-MRI can be used as an early marker of effective tumor response to choline targeted therapies. DW-MRI combined to choline spectroscopy may provide a useful non-invasive marker for the early clinical assessment of tumor response to therapies targeting choline signaling.

Sensitive simultaneous measurements of oxygenation and extracellular pH by EPR using a stable monophosphonated trityl radical and lithium phthalocyanine.

The monitoring of acidosis and hypoxia is crucial because both factors promote cancer progression and impact the efficacy of anti-cancer treatments. A phosphonated tetrathiatriarylmethyl (pTAM) has been previously described to monitor both parameters simultaneously, but the sensitivity to tackle subtle changes in oxygenation was limited. Here, we describe an innovative approach combining the pTAM radical and lithium phthalocyanine (LiPc) crystals to provide sensitive simultaneous measurements of extracellular pH (pHe) and pO2. Both parameters can be measured simultaneously as both EPR spectra do not overlap, with a gain in sensitivity to pO2 variations by a factor of 10. This procedure was applied to characterize the impact of carbogen breathing in a breast cancer 4T1 model as a proof-of-concept. No significant change in pHe and pO2 was observed using pTAM alone, while LiPc detected a significant increase in tumor oxygenation. Interestingly, we observed that pTAM systematically overestimated the pO2 compared to LiPc. In addition, we analyzed the impact of an inhibitor (UK-5099) of the mitochondrial pyruvate carrier (MPC) on the tumor microenvironment. In vitro, the exposure of 4T1 cells to UK-5099 for 24 h induced a decrease in pHe and oxygen consumption rate (OCR). In vivo, a significant decrease in tumor pHe was observed in UK-5099-treated mice, while there was no change for mice treated with the vehicle. Despite the change observed in OCR, no significant change in tumor oxygenation was observed after the UK-5099 treatment. This approach is promising for assessing in vivo the effect of treatments targeting tumor metabolism.

Highly Sensitive Detection of Melanin in Melanomas Using Multi-harmonic Low Frequency EPR.

PURPOSE: Low frequency EPR can noninvasively detect endogenous free radical melanin in melanocytic skin lesions and could potentially discriminate between benign atypical nevi and malignant melanoma lesions. We recently succeeded in demonstrating the ability of clinical EPR to noninvasively detect the endogenous melanin free radical in skin lesions of patients. However, the signal-to-noise ratio (SNR) was extremely low warranting further research to boost the sensitivity of detection. In the present study, we assessed the performance of a clinical EPR system with the capability to perform multi-harmonic (MH) analysis for the detection of melanin. PROCEDURES: The sensitivity of MH-EPR was compared with a classical continuous wave (CW)-EPR (1st harmonic) detection in vitro in melanin phantoms, in vivo in melanoma models with cells implanted in the skin, in lymph nodes and having colonized the lungs, and finally on phantoms placed at the surface of human skin. RESULTS: In vitro, we observed an increase in SNR by a factor of 10 in flat melanin phantoms when using MH analysis compared to CW combined with an increase in modulation amplitude. In B16 melanomas having grown in the skin of hairless mice, we observed a boost in sensitivity in vivo similar to that observed in vitro with the capability to detect melanoma cells at an earlier stage of development. MH-EPR was also able to detect non-invasively the melanin signal coming from melanoma cells present in lymph nodes as well as in lungs. We also observed a boost of sensitivity using phantoms of melanin placed at the surface of human skin. CONCLUSIONS: Overall, our results are paving the way for new clinical trials that will use MH clinical EPR for the characterization of pigmented skin lesions.

Hexafluorobenzene in comparison with perfluoro-15-crown-5-ether for repeated monitoring of oxygenation using 19F MRI in a mouse model.

Hexafluorobenzene (HFB) and perfluoro-15-crown-5-ether (15C5) were compared as fluorine reporter probes of tissue oxygenation using (19)F MRI for dynamic assessment of muscle oxygenation, with special focus on muscle tissue toxicity of the probes, and consecutive alteration of animal behavior. The latter were also compared in terms of sensitivity to changes in oxygenation as well as of signal-to-noise ratio for accurate pO(2) measurements. For that purpose, mouse muscles were imaged at 11.7 T, at 2- and 36-h after intramuscular injection of HFB or 15C5. Histological analysis of the muscle tissue revealed a lack of toxicity for 15C5 from 2 up to 36-h postinjection, whereas HFB induced tissue necrosis, blood clots and thrombosis as soon as 24-h postinjection. This muscle toxicity led to a limitation in mice mobility 24-h after injection of HFB as evidenced by behavioral testing (open-field, grip strength, and catwalk tests), which was not the case after 15C5 intramuscular injection. Finally, pO(2) measurements assessed 2-h postinjection showed consistent values with both probes, evidencing cross-validation of the (19)F MRI oximetry technique for acute measurements. However, the measurement at 36-h was hampered for HFB, which showed significant lower values of muscle pO(2), whereas 15C5 was able to reliably assess muscle pO(2) at 36-h postinjection.

Mapping of oxygen by imaging lipids relaxation enhancement: a potential sensitive endogenous MRI contrast to map variations in tissue oxygenation.

PURPOSE: Because of its paramagnetic properties, oxygen may act as an endogenous magnetic resonance imaging contrast agent by changing proton relaxation rates. Changes in tissue oxygen concentrations have been shown to produce changes in relaxation rate R1 of water. The aim of the study was to improve the sensitivity of oxygen enhanced R1 imaging by exploiting the higher solubility of oxygen in lipids (as compared with water) to sensitively monitor changes in tissue oxygen levels by selectively measuring the R1 of lipids. METHODS: The method, with the acronym "MOBILE" (mapping of oxygen by imaging lipids relaxation enhancement), was applied in different mouse models of hypoxic processes on a 11.7 T magnetic resonance imaging system. MOBILE was compared with R*2, R1 of water, and with pO2 measurements (using electron paramagnetic resonance oximetry). MOBILE was also applied in the brain of healthy human volunteers exposed to an oxygen breathing challenge on a 3 T magnetic resonance imaging system. RESULTS: MOBILE was shown to be able to monitor changes in oxygenation in tumor, peripheral, liver, and brain tissues. The clinical translation was demonstrated in human volunteers. CONCLUSION: MOBILE arises as a promising noninvasive and sensitive tool for diagnosis and therapeutic guidance in disorders involving hypoxia.

Application of MOBILE (mapping of oxygen by imaging lipids relaxation enhancement) to study variations in tumor oxygenation.

The aim of the study was to sensitively monitor changes in tumor oxygen using the MOBILE (mapping of oxygen by imaging lipids relaxation enhancement) technique. This method was applied in mammary tumor mouse models on an 11.7T Bruker MRI system. MOBILE was compared with functional imaging R2*, R1 of water and with pO2 measurements (using EPR oximetry and O2-dependent fluorescence quenching measurements). MOBILE was shown to be capable to monitor changes in oxygenation in tumor tissues.

Towards Characterization of Skin Melanoma in the Clinic by Electron Paramagnetic Resonance (EPR) Spectroscopy and Imaging of Melanin.

The incidence of melanoma is continuously increasing over time. Melanoma is the most aggressive skin cancer, significantly reducing quality of life and survival rates of patients at advanced stages. Therefore, early diagnosis remains the key to change the prognosis of patients with melanoma. In this context, advanced technologies are under evaluation to increase the accuracy of the diagnostic, to better characterize the lesions and visualize their possible invasiveness in the epidermis. Among the innovative methods, because melanin is paramagnetic, clinical low frequency electron paramagnetic resonance (EPR) that characterizes the melanin content in the lesion has the potential to be an adjunct diagnostic method of melanoma. In this review, we first summarize the challenges faced by dermatologists and oncologists in melanoma diagnostic and management. We also provide a historical perspective on melanin detection with a focus on EPR spectroscopy/imaging of melanomas. We describe key elements that allow EPR to move from in vitro studies to in vivo and finally to patients for melanoma studies. Finally, we provide a critical view on challenges to meet to make EPR operational in the clinic to characterize pigmented lesions.

Metformin improves cancer immunotherapy by directly rescuing tumor-infiltrating CD8 T lymphocytes from hypoxia-induced immunosuppression.

BACKGROUND: Despite their revolutionary success in cancer treatment over the last decades, immunotherapies encounter limitations in certain tumor types and patients. The efficacy of immunotherapies depends on tumor antigen-specific CD8 T-cell viability and functionality within the immunosuppressive tumor microenvironment, where oxygen levels are often low. Hypoxia can reduce CD8 T-cell fitness in several ways and CD8 T cells are mostly excluded from hypoxic tumor regions. Given the challenges to achieve durable reduction of hypoxia in the clinic, ameliorating CD8 T-cell survival and effector function in hypoxic condition could improve tumor response to immunotherapies. METHODS: Activated CD8 T cells were exposed to hypoxia and metformin and analyzed by fluorescence-activated cell sorting for cell proliferation, apoptosis and phenotype. In vivo, metformin was administered to mice bearing hypoxic tumors and receiving either adoptive cell therapy with tumor-specific CD8 T cells, or immune checkpoint inhibitors; tumor growth was followed over time and CD8 T-cell infiltration, survival and localization in normoxic or hypoxic tumor regions were assessed by flow cytometry and immunofluorescence. Tumor oxygenation and hypoxia were measured by electron paramagnetic resonance and pimonidazole staining, respectively. RESULTS: We found that the antidiabetic drug metformin directly improved CD8 T-cell fitness in hypoxia, both in vitro and in vivo. Metformin rescued murine and human CD8 T cells from hypoxia-induced apoptosis and increased their proliferation and cytokine production, while blunting the upregulation of programmed cell death protein 1 and lymphocyte-activation gene 3. This appeared to result from a reduced production of reactive oxygen species, due to the inhibition of mitochondrial complex I. Differently from what others reported, metformin did not reduce tumor hypoxia, but rather increased CD8 T-cell infiltration and survival in hypoxic tumor areas, and synergized with cyclophosphamide to enhance tumor response to adoptive cell therapy or immune checkpoint blockade in different tumor models. CONCLUSIONS: This study describes a novel mechanism of action of metformin and presents a promising strategy to achieve immune rejection in hypoxic and immunosuppressive tumors, which would otherwise be resistant to immunotherapy.

Evaluation of Syrosingopine, an MCT Inhibitor, as Potential Modulator of Tumor Metabolism and Extracellular Acidification.

Extracellular acidification has been shown to be an important characteristic of invasive tumors, as it promotes invasion and migration but also resistance to treatments. Targeting transporters involved in the regulation of tumor pH constitutes a promising anti-tumor approach, as it would disrupt cellular pH homeostasis and negatively impact tumor growth. In this study, we evaluated the impact of syrosingopine, an inhibitor of MCT1 and MCT4, as a modulator of tumor metabolism and extracellular acidification in human breast cancer (MDA-MB-231) and pharyngeal squamous cell carcinoma (FaDu) cell models. In both models in vitro, we observed that exposure to syrosingopine led to a decrease in the extracellular acidification rate, intracellular pH, glucose consumption, lactate secretion and tumor cell proliferation with an increase in the number of late apoptotic/necrotic cells. However, in vivo experiments using the MDA-MB-231 model treated with a daily injection of syrosingopine did not reveal any significant change in extracellular pH (pHe) (as measured using CEST-MRI) or primary tumor growth. Overall, our study suggests that targeting MCT could lead to profound changes in tumor cell metabolism and proliferation, and it warrants further research to identify candidates without off-target effects.

Statins Alleviate Tumor Hypoxia in Prostate Cancer Models by Decreasing Oxygen Consumption: An Opportunity for Radiosensitization?

BACKGROUND: Because statins were found to decrease the oxygen consumption rate (OCR) of a variety of normal cells, our hypothesis was that statins may also decrease the OCR of cancer cells, alleviate tumor hypoxia and radiosensitize tumors. METHODS: OCR was assessed using the Seahorse XF96 technology and EPR respirometry in PC-3 prostate cancer cells. Mitochondrial superoxide production was measured by EPR with mitoTEMPO-H as a sensing probe. Tumor pO2 was measured in vivo using low-frequency EPR oximetry to define the optimal window of reoxygenation, the time at which tumors were irradiated with a single 6 Gy dose with a Cesium-137 irradiator. RESULTS: 24-h exposure to simvastatin and fluvastatin significantly decreased the OCR of PC-3 cancer cells. An increase in mitochondrial superoxide levels was also observed after fluvastatin exposure. The PC-3 prostate cancer model was found highly hypoxic at the basal level. When mice were treated with simvastatin or fluvastatin (daily injection of 20 mg/kg), tumor oxygenation increased 48 and 72 h after initiation of the treatment. However, despite reoxygenation, simvastatin did not sensitize the PC-3 tumor model to RT. CONCLUSIONS: exposure to statins affect tumor metabolism and tumor oxygenation, however, with limited impact on tumor growth with or without irradiation.

Tumor Metabolism Is Affected by Obesity in Preclinical Models of Triple-Negative Breast Cancer.

Obesity is characterized by an excessive fat mass accumulation associated with multiple disorders, including impaired glucose homeostasis, altered adipokine levels, and hyperlipidemia. Despite clear associations between tumor progression and obesity, the effects of these disorders on tumor metabolism remain largely unknown. Thus, we studied the metabolic differences between tumors of obese and lean mice in murine models of triple-negative breast cancer (E0771 and PY8819). For this purpose, a real-time hyperpolarized 1-13C-pyruvate-to-lactate conversion was studied before and after glucose administration in fasting mice. This work was completed by U-13C glucose tracing experiments using nuclear magnetic resonance (NMR) spectroscopy, as well as mass spectrometry (MS). Ex vivo analyses included immunostainings of major lipid, glucose, and monocarboxylic acids transporters. On the one hand, we discovered that tumors of obese mice yield higher lactate/pyruvate ratios after glucose administration. On the other hand, we found that the same tumors produce higher levels of lactate and alanine from glucose than tumors from lean mice, while no differences on the expression of key transporters associated with glycolysis (i.e., GLUT1, MCT1, MCT4) have been observed. In conclusion, our data suggests that breast tumor metabolism is regulated by the host's physiological status, such as obesity and diabetes.

Combined HP 13C Pyruvate and 13C-Glucose Fluxomic as a Potential Marker of Response to Targeted Therapies in YUMM1.7 Melanoma Xenografts.

A vast majority of BRAF V600E mutated melanoma patients will develop resistance to combined BRAF/MEK inhibition after initial clinical response. Resistance to targeted therapy is described to be accompanied by specific metabolic changes in melanoma. The aim of this work was to evaluate metabolic imaging using 13C-MRS (Magnetic Resonance Spectroscopy) as a marker of response to BRAF/MEK inhibition in a syngeneic melanoma model. Tumor growth was significantly delayed in mice bearing YUMM1.7 melanoma xenografts treated with the BRAF inhibitor vemurafenib, and/or with the MEK inhibitor trametinib, in comparison with the control group. 13C-MRS was performed in vivo after injection of hyperpolarized (HP) 13C-pyruvate, at baseline and 24 h after treatment, to evaluate dynamic changes in pyruvate-lactate exchange. Furthermore, ex vivo 13C-MRS steady state metabolic tracing experiments were performed after U-13C-glucose or 5-13C-glutamine injection, 24 h after treatment. The HP 13C-lactate-to-pyruvate ratio was not modified in response to BRAF/MEK inhibition, whereas the production of 13C-lactate from 13C-glucose was significantly reduced 24 h after treatment with vemurafenib, trametinib, or with the combined inhibitors. Conversely, 13C-glutamine metabolism was not modified in response to BRAF/MEK inhibition. In conclusion, we identified 13C-glucose fluxomic as a potential marker of response to BRAF/MEK inhibition in YUMM1.7 melanoma xenografts.

Noninvasive detection of the endogenous free radical melanin in human skin melanomas using electron paramagnetic resonance (EPR).

We explored the capability of low-frequency Electron Paramagnetic Resonance (EPR) to noninvasively detect melanin (a stable semiquinone free radical) in the human skin. As previous in vitro studies on biopsies suggested that the EPR signal from melanin was different when measured in skin melanomas or benign nevi, we conducted a prospective first-in-man clinical EPR study in patients with skin lesions suspicious of melanoma. EPR spectra were obtained using a spectrometer operating at 1 GHz, with a surface coil placed over the area of interest. Two clinical studies were carried out: 1) healthy volunteers (n = 45) presenting different skin phototypes; 2) patients (n = 88) with skin lesions suspicious of melanoma (n = 100) requiring surgical resection. EPR data obtained before surgery were compared with histopathology results. The method was not sensitive enough to measure differences in melanin content due to changes in skin pigmentation. In patients, 92% of the spectra were analyzable. The EPR signal of melanin was significantly higher (p < 0.0001) in melanoma lesions (n = 26) than that in benign atypical nevi (n = 62). A trend toward a higher signal intensity (though not significant) was observed in high Breslow depth melanomas (a marker of skin invasion) than in low Breslow lesions. To date, no naturally occurring free radicals have been detected by low-frequency EPR systems adapted for clinical studies. Here, we demonstrated for the first time the ability of this technology to detect an endogenous free radical, opening new avenues for evaluating clinical EPR as a potential aid in the diagnosis of pigmented skin lesions.

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer.

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.