Lactate Depletion Research Articles

Sono‐Triggered Cascade Lactate Depletion by Semiconducting Polymer Nanoreactors for Cuproptosis‐Immunotherapy of Pancreatic Cancer

AbstractThe high level of lactate in tumor microenvironment not only promotes tumor development and metastasis, but also induces immune escape, which often leads to failures of various tumor therapy strategies. We here report a sono‐triggered cascade lactate depletion strategy by using semiconducting polymer nanoreactors (SPNLCu) for cancer cuproptosis‐immunotherapy. The SPNLCu mainly contain a semiconducting polymer as sonosensitizer, lactate oxidase (LOx) conjugated via a reactive oxygen species (ROS)‐cleavable linker and chelated Cu2+. Upon ultrasound (US) irradiation, the semiconducting polymer generates singlet oxygen (1O2) to cut ROS‐cleavable linker to allow the release of LOx that catalyzes lactate depletion to produce hydrogen peroxide (H2O2). The Cu2+ will be reduced to Cu+ in tumor microenvironment, which reacts with the produced H2O2 to obtain hydroxyl radical (⋅OH) that further improves LOx release via destroying ROS‐cleavable linkers. As such, sono‐triggered cascade release of LOx achieves effective lactate depletion, thus relieving immunosuppressive roles of lactate. Moreover, the toxic Cu+ induces cuproptosis to cause immunogenic cell death (ICD) for activating antitumor immunological effect. SPNLCu are used to treat both subcutaneous and deep‐tissue orthotopic pancreatic cancer with observably enhanced efficacy in restricting the tumor growths. This study thus provides a precise and effective lactate depletion tactic for cancer therapy.

Sono-Triggered Cascade Lactate Depletion by Semiconducting Polymer Nanoreactors for Cuproptosis-Immunotherapy of Pancreatic Cancer.

The high level of lactate in tumor microenvironment not only promotes tumor development and metastasis, but also induces immune escape, which often leads to failures of various tumor therapy strategies. We here report a sono-triggered cascade lactate depletion strategy by using semiconducting polymer nanoreactors (SPNLCu) for cancer cuproptosis-immunotherapy. The SPNLCu mainly contain a semiconducting polymer as sonosensitizer, lactate oxidase (LOx) conjugated via a reactive oxygen species (ROS)-cleavable linker and chelated Cu2+. Upon ultrasound (US) irradiation, the semiconducting polymer generates singlet oxygen (1O2) to cut ROS-cleavable linker to allow the release of LOx that catalyzes lactate depletion to produce hydrogen peroxide (H2O2). The Cu2+ will be reduced to Cu+ in tumor microenvironment, which reacts with the produced H2O2 to obtain hydroxyl radical (⋅OH) that further improves LOx release via destroying ROS-cleavable linkers. As such, sono-triggered cascade release of LOx achieves effective lactate depletion, thus relieving immunosuppressive roles of lactate. Moreover, the toxic Cu+ induces cuproptosis to cause immunogenic cell death (ICD) for activating antitumor immunological effect. SPNLCu are used to treat both subcutaneous and deep-tissue orthotopic pancreatic cancer with observably enhanced efficacy in restricting the tumor growths. This study thus provides a precise and effective lactate depletion tactic for cancer therapy.

A Lactate-Depleting metal organic framework-based nanocatalyst reinforces intratumoral T cell response to boost anti-PD1 immunotherapy

Infiltration and activation of intratumoral T lymphocytes are critical for immune checkpoint blockade (ICB) therapy. Unfortunately, the low tumor immunogenicity and immunosuppressive tumor microenvironment (TME) induced by tumor metabolic reprogramming cooperatively hinder the ICB efficacy. Herein, we engineered a lactate-depleting MOF-based catalytic nanoplatform (LOX@ZIF-8@MPN), encapsulating lactate oxidase (LOX) within zeolitic imidazolate framework-8 (ZIF-8) coupled with a coating of metal polyphenol network (MPN) to reinforce T cell response based on a “two birds with one stone” strategy. LOX could catalyze the degradation of the immunosuppressive lactate to promote vascular normalization, facilitating T cell infiltration. On the other hand, hydrogen peroxide (H2O2) produced during lactate depletion can be transformed into anti-tumor hydroxyl radical (•OH) by the autocatalytic MPN-based Fenton nanosystem to trigger immunogenic cell death (ICD), which largely improved the tumor immunogenicity. The combination of ICD and vascular normalization presents a better synergistic immunopotentiation with anti-PD1, inducing robust anti-tumor immunity in primary tumors and recurrent malignancies. Collectively, our results demonstrate that the concurrent depletion of lactate to reverse the immunosuppressive TME and utilization of the by-product from lactate degradation via cascade catalysis promotes T cell response and thus improves the effectiveness of ICB therapy.

Intratumoral Lactate Depletion Based on Injectable Nanoparticles-Hydrogel Composite System Synergizes with Immunotherapy against Postablative Hepatocellular Carcinoma Recurrence.

Thermal ablation is a crucial therapeutic modality for hepatocellular carcinoma (HCC), but its efficacy is often hindered by the high recurrence rate attributed to insufficient ablation. Furthermore, the residual tumors following insufficient ablation exhibit a more pronounced immunosuppressive state, which accelerates the disease progression and leads to immune checkpoint blockade (ICB) resistance. Herein, evidence is presented that heightened intratumoral lactate accumulation, stemming from the augmented glycolytic activity of postablative residual HCC cells, may serve as a crucial driving force in exacerbating the immunosuppressive state of the tumor microenvironment (TME). To address this, an injectable nanoparticles-hydrogel composite system (LOX-MnO2 @Gel) is designed that gradually releases lactate oxidase (LOX)-loaded hollow mesoporous MnO2 nanoparticles at the tumor site to continuously deplete intratumoral lactate via a cascade catalytic reaction. Using subcutaneous and orthotopic HCC tumor-bearing mouse models, it is confirmed that LOX-MnO2 @Gel-mediated local lactate depletion can transform the immunosuppressive postablative TME into an immunocompetent one and synergizes with ICB therapy to significantly inhibit residual HCC growth and lung metastasis, thereby prolonging the survival of mice postablation. The work proposes an appealing strategy for synergistically combining antitumor metabolic therapy with immunotherapy to combat postablative HCC recurrence.

In vivo calibration of genetically encoded metabolite biosensors must account for metabolite metabolism during calibration and cellular volume.

Isotopic assays of brain glucose utilization rates have been used for more than four decades to establish relationships between energetics, functional activity, and neurotransmitter cycling. Limitations of these methods include the relatively long time (1-60 min) for the determination of labeled metabolite levels and the lack of cellular resolution. Identification and quantification of fuels for neurons and astrocytes that support activation and higher brain functions are a major, unresolved issues. Glycolysis is preferentially up-regulated during activation even though oxygen level and supply are adequate, causing lactate concentrations to quickly rise during alerting, sensory processing, cognitive tasks, and memory consolidation. However, the fate of lactate (rapid release from brain or cell-cell shuttling coupled with local oxidation) is long disputed. Genetically encoded biosensors can determine intracellular metabolite concentrations and report real-time lactate level responses to sensory, behavioral, and biochemical challenges at the cellular level. Kinetics and time courses of cellular lactate concentration changes are informative, but accurate biosensor calibration is required for quantitative comparisons of lactate levels in astrocytes and neurons. An in vivo calibration procedure for the Laconic lactate biosensor involves intracellular lactate depletion by intravenous pyruvate-mediated trans-acceleration of lactate efflux followed by sensor saturation by intravenous infusion of high doses of lactate plus ammonium chloride. In the present paper, the validity of this procedure is questioned because rapid lactate-pyruvate interconversion in blood, preferential neuronal oxidation of both monocarboxylates, on-going glycolytic metabolism, and cellular volumes were not taken into account. Calibration pitfalls for the Laconic lactate biosensor also apply to other metabolite biosensors that are standardized in vivo by infusion of substrates that can be metabolized in peripheral tissues. We discuss how technical shortcomings negate the conclusion that Laconic sensor calibrations support the existence of an in vivo astrocyte-neuron lactate concentration gradient linked to lactate shuttling from astrocytes to neurons to fuel neuronal activity.

Mesothelin targeted nano-system enhanced chemodynamic therapy and tirapazamine chemotherapy via lactate depletion

Mesothelin targeted nano-system enhanced chemodynamic therapy and tirapazamine chemotherapy via lactate depletion

O2-Supplying Nanozymes Alleviate Hypoxia and Deplete Lactate to Eliminate Tumors and Activate Antitumor Immunity

Direct hypoxia alleviation and lactate depletion in the tumor microenvironment (TME) are promising for effective cancer therapy but still very challenging. To address this challenge, the current research directly reshapes the TME for inhibiting tumor growth and activating the antitumor immunity using a drug-free nanozyme. Herein, the acid-sensitive nanozymes were constructed based on peroxidized layered double hydroxide nanoparticles for O2 self-supply and self-boosted lactate depletion. The coloading of partially cross-linked catalase and lactate oxidase enabled the acid-sensitive nanozymes to promote three reactions, that is, (1) H2O2 generation from MgO2 hydrolysis (30% at pH 7.4 vs 63% at pH 6.0 in 8 h); (2) O2 generation from H2O2 (12% at pH 7.4 vs 21% at pH 6.0 in 2 h); and (3) lactate depletion by in situ generated O2 (50% under hypoxia vs 75% under normoxia in 24 h in vitro) in parallel or tandem. These promoted reactions together efficiently induced colon cancer cell apoptosis under the hypoxic conditions, significantly inhibited tumor growth (>95%), and suppressed distant tumor growth upon seven administrations in every 3 days and moreover transformed the immunosuppressive tumor into "hot" one in the colon tumor-bearing mouse model. This is the first example for a nanozyme that supplies sufficient O2 for hypoxia relief and lactate depletion, thus providing a new insight into drug-free nanomaterial-mediated TME-targeted cancer therapy.

Insights into the Effect of Catalytic Intratumoral Lactate Depletion on Metabolic Reprogramming and Immune Activation for Antitumoral Activity

Lactate, a characteristic metabolite of the tumor microenvironment (TME), drives immunosuppression and promotes tumor progression. Material-engineered strategies for intratumoral lactate modulations demonstrate their promise for tumor immunotherapy. However, understanding of the inherent interconnections of material-enabled lactate regulation, metabolism, and immunity in the TME is scarce. To address this issue, urchin-like catalysts of the encapsulated Gd-doped CeO2 , syrosingopine, and lactate oxidase are used in ZIF-8 (USL, where U, S, and L represent the urchin-like Gd-doped CeO2 @ZIF-8, syrosingopine, and lactate oxidase, respectively) and orthotopic tumor models. The instructive relationships of intratumoral lactate depletion, metabolic reprogramming, and immune activation for catalytic immunotherapy of tumors is illustrated. The catalysts efficiently oxidize intratumoral lactate and significantly promote tumor cell apoptosis by in situ-generated ·OH, thereby reducing glucose supply and inducing mitochondrial damage via lactate depletion, thus reprogramming glycometabolism. Subsequently, such catalytic metabolic reprogramming evokes both local and systemic antitumor immunity by activating M1-polarizaed macrophages and CD8+ T cells, leading to potent antitumor immunity. This study provides valuable mechanistic insights into material-interfered tumor therapy through intratumoral lactate depletion and consequential connection with metabolic reprogramming and immunity remodeling, which is thought to enhance the efficacy of immunotherapy.

SnSe Nanosheets Mimic Lactate Dehydrogenase to Reverse Tumor Acid Microenvironment Metabolism for Enhancement of Tumor Therapy.

The acidic tumor microenvironment (TME) is unfriendly to the activity and function of immune cells in the TME. Here, we report inorganic nanozymes (i.e., SnSe NSs) that mimic the catalytic activity of lactate dehydrogenase to degrade lactate to pyruvate, contributing to the metabolic treatment of tumors. As found in this study, SnSe NSs successfully decreased lactate levels in cells and tumors, as well as reduced tumor acidity. This is associated with activation of the immune response of T cells, thus alleviating the immunosuppressive environment of the TME. More importantly, the nanozyme successfully inhibited tumor growth in mutilate mouse tumor models. Thus, SnSe NSs show a promising result in lactate depletion and tumor suppression, which exemplifies its potential strategy in targeting lactate for metabolic therapy.

Cascades catalyzed lactic acid depletion coordinated with photothermal therapy for enhanced cancer immunotherapy

A large amount of lactate accumulated in the tumor tissues plays an important role in tumor angiogenesis, recurrence, metastasis, drug resistance and immunosuppression. However, there is a lack of a simple and efficient lactate depletion strategy. Herein, a red blood cell membrane (mRBC)-camouflaged polydopamine (PDA)-coated dendritic mesoporous silicon nanoparticle (DMSN) was constructed for the delivery of lactate oxidase (LOX) to achieve coordinated catalytic consumption of intracellular and extracellular lactate and modulation of immunotherapy (denoted as DPLM). Due to the encapsulation of the mRBC, the DPLM could achieve long circulation in the blood and gradually accumulate into the tumor tissues within 8 h. The PDA which possessed catalase-like nature could catalyze the decomposition reaction of H2O2 in hypoxic tumor tissue, therefore providing oxygen for LOX to oxidize extracellular lactate. Meanwhile, with the help of the photothermal conversion ability of PDA, the laser-trigged low-temperature photothermal effect on DPLM could cause the endosomal escape, which facilitated the delivery of LOX into cytoplasm to deplete intracellular lactate. In addition, due to the photothermal effect, tumor cells could release immune signals that promoted immune cells infiltration into tumor tissues. This study provides an innovative LOX delivery system to regulate tumor acid environment and relieve immunosuppression.

Dual Closed-Loop of Catalyzed Lactate Depletion and Immune Response to Potentiate Photothermal Immunotherapy.

Lactate accumulation in the solid tumor is highly relevant to the immunosuppressive tumor microenvironment (TME). Targeting lactate metabolism significantly enhances the efficacy of immunotherapy. However, lactate depletion by lactate oxidase (LOX) consumes oxygen and results in the aggravated hypoxia situation, counteracting the benefit of lactate depletion. Beyond the TME regulation, it is necessary to initiate the effective immunity cycle for therapeutic purposes. In this fashion, dual close-loop of catalyzed lactate depletion and immune response by a rational material design are established to address this issue. Here, we constructed PEG-modified mesoporous polydopamine nanoparticles with Cu2+ chelation and LOX encapsulation (denoted as mCuLP). After mCuLP nanosystems targeting into the tumor sites, released LOX consumes lactate to H2O2. Subsequently, the produced H2O2 is further catalyzed by Cu2+-chelated mPDA to produce oxygen, supplying the oxygen source for the closed-loop of lactate depletion. Meanwhile, the mild PTT caused by the photothermal mPDA induces ICD of tumor cells to promote DC maturation and then T lymphocyte infiltration to kill tumor cells, which forms another closed-loop for cancer immunity. Therefore, this dual closed-loop strategy of mCuLP nanosystems effectively inhibits tumor growth, providing a promising treatment modality to cancer immunotherapy.

Loss-of-Function Genetic Screening Identifies Aldolase A as an Essential Driver for Liver Cancer Cell Growth Under Hypoxia.

Background and AimsHypoxia is a common feature of the tumor microenvironment (TME), which promotes tumor progression, metastasis, and therapeutic drug resistance through a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of HCC.Approach and ResultsFrom a genome‐wide CRISPR/CRISPR‐associated 9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion and consequently inhibits tumor growth. Supplementation with lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by hypoxia‐inducible factor‐1α and fat mass and obesity–associated protein–mediated N6‐methyladenosine modification through transcriptional and posttranscriptional regulation, respectively. Analysis of The Cancer Genome Atlas shows that elevated levels of ALDOA are significantly correlated with poor prognosis of patients with HCC. In a screen of Food and Drug Administration–approved drugs based on structured hierarchical virtual platforms, we identified the sulfamonomethoxine derivative compound 5 (cpd‐5) as a potential inhibitor to target ALDOA, evidenced by the antitumor activity of cpd‐5 in preclinical patient‐derived xenograft models of HCC.ConclusionsOur work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in the hypoxic TME is a promising therapeutic strategy for treating HCC.

Potential therapeutics using tumor-secreted lactate in nonsmall cell lung cancer

Targeted-therapy failure in treating nonsmall cell lung cancer (NSCLC) frequently occurs because of the emergence of drug resistance and genetic mutations. The same mutations also result in aerobic glycolysis, which further antagonizes outcomes by localized increases in lactate, an immune suppressor. Recent evidence indicates that enzymatic lowering of lactate can promote an oncolytic immune microenvironment within the tumour. Here, we review factors relating to lactate expression in NSCLC and the utility of lactate oxidase (LOX) for governing therapeutic delivery, its role in lactate oxidation and turnover, and relationships between lactate depletion and immune cell populations. The lactate-rich characteristic of NSCLC provides an exploitable property to potentially improve NSCLC outcomes and design new therapeutic strategies to integrate with conventional therapies.

Lactate Consumption via Cascaded Enzymes Combined VEGF siRNA for Synergistic Anti‐Proliferation and Anti‐Angiogenesis Therapy of Tumors

Lactate, as the most abundant component with concentrations of 4-40mm in tumors, contributes to the regulation of metabolic pathways, angiogenesis, and immunosuppression, exhibiting remarkable potential in cancer treatment. Therefore, a codelivery strategy that combined the cascaded enzymes Lactate oxidase/Catalase (LOx/CAT) and vascular endothelial growth factor (VEGF) siRNA (siVEGF) to suppress tumor proliferation and angiogenesis synergistically is creatively proposed. In brief, the cationic liposomes (LIP) encapsulated with LOx/CAT and siVEGF via hydrophilic interaction and electrostatic adsorption followed by coating with PEGylated phenylboronic acid (PP) is established (PPL@[LOX+CAT]). Moreover, a simple 3-aminophenylboronic acid (PBA)-shielded strategy via fructose (Fru) is applied to further enhance the targeting efficiency in the tumor site. The obtained co-encapsulated nanoparticles (NPs) can simultaneous intracellular release of LOx/CAT and siVEGF, and the collaborative use of LOx and CAT can promote lactate consumption even under a hypoxic tumor microenvironment (TME) without producing systemic toxicity. The combined application of lactate depletion and VEGF silencing demonstrated the efficient migration suppression of 4T1 cells in vitro and superior antitumor and antimetastatic properties in vivo. This work offers a promising tumor treatment strategy via integrating cascaded enzymes and gene therapy, and explores a promising therapy regimen for 4T1 triple-negative breast cancer.

Openwork@Dendritic Mesoporous Silica Nanoparticles for Lactate Depletion and Tumor Microenvironment Regulation.

The direct depletion of lactate accumulated in the tumor microenvironment holds promise for cancer therapy but remains challenging. Herein, we report a one-pot synthesis of openwork@ dendritic mesoporous silica nanoparticles (ODMSNs) to address this problem. ODMSNs self-assembled through a time-resolved lamellar growth mechanism feature an openworked core and a dendritic shell, both constructed by silica nanosheets of ≈3 nm. With a large pore size, high surface area and pore volume, ODMSNs exhibited a high loading capacity (>0.7 g g-1 ) of lactate oxidase (LOX) and enabled intratumoral lactate depletion by >99.9 %, leading to anti-angiogenesis, down-regulation of vascular endothelial growth factor, and increased tumor hypoxia. The latter event facilitates the activation of a co-delivered prodrug for enhancing anti-tumor and anti-metastasis efficacy. This study provides an innovative nano-delivery system and demonstrates the first example of direct lactate-depletion-enabled chemotherapy.

Openwork@Dendritic Mesoporous Silica Nanoparticles for Lactate Depletion and Tumor Microenvironment Regulation

AbstractThe direct depletion of lactate accumulated in the tumor microenvironment holds promise for cancer therapy but remains challenging. Herein, we report a one‐pot synthesis of openwork@ dendritic mesoporous silica nanoparticles (ODMSNs) to address this problem. ODMSNs self‐assembled through a time‐resolved lamellar growth mechanism feature an openworked core and a dendritic shell, both constructed by silica nanosheets of ≈3 nm. With a large pore size, high surface area and pore volume, ODMSNs exhibited a high loading capacity (>0.7 g g−1) of lactate oxidase (LOX) and enabled intratumoral lactate depletion by >99.9 %, leading to anti‐angiogenesis, down‐regulation of vascular endothelial growth factor, and increased tumor hypoxia. The latter event facilitates the activation of a co‐delivered prodrug for enhancing anti‐tumor and anti‐metastasis efficacy. This study provides an innovative nano‐delivery system and demonstrates the first example of direct lactate‐depletion‐enabled chemotherapy.

Repolarization of M2 to M1 Macrophages Triggered by Lactate Oxidase Released from Methylcellulose Hydrogel.

Deregulated proliferation of tumors is generally associated with altered energy metabolism. A high rate of anaerobic glycolysis in solid tumors contributes to an acidification of pH to ∼6.7-7.2 in the tumor microenvironment and lactate accumulation. Macrophages in the tumor microenvironment can be educated by tumor cells. Tumor-derived lactate induces the polarization of M2 macrophages and promotes tumor invasion and metastasis. However, a particular challenge is to sustain lactate depletion. We propose that the repolarization of the tumor-supportive M2 macrophage to the tumor-suppressive M1 macrophage after the depletion of lactate by lactate oxidase (LOX) released from the hydrogels in the tumor microenvironment may enhance the antitumor treatment efficacy.

Monocarboxylate transporter 1 is a key player in glioma-endothelial cell crosstalk.

Glioblastoma (GBM) is one of the most glycolytic and angiogenic human tumors, characteristics that contribute to the poor prognosis associated with this type of tumor. A lactate shuttle has been described between tumor cells and endothelial cells (ECs), with the monocarboxylate transporters (MCTs) acting as important players in this tumor-EC communication. In this study, we aimed to understand how the tumor microenvironment modulates EC metabolism, and to characterize the role of MCTs in the glioma-brain EC crosstalk. Exposure of human brain microvascular ECs (HBMEC) to GBM cell-conditioned media increased the expression of MCT1, which corresponded to activation of oxidative metabolism and an increase in angiogenic capacity, as determined by increased proliferation, migration, and vessel assembly. Lactate depletion from the microenvironment or inhibition of lactate uptake in HBMEC induced an increase in lactate production and a decrease in proliferation, migration, and vessel assembly. Moreover, addition of lactate to HBMEC media promoted activation of AKT and AMPK pathways and increased expression in NFκB, HIF-1α, and the lactate receptor GPR81. Here, we demonstrate a role for MCT1 as a mediator of lactate signaling between glioma cells and brain ECs. Our results suggest that MCT1 can mediate EC metabolic reprograming, proliferation, and vessel sprouting in response to tumor signaling. Thus, targeting MCT1 in both tumor cells and brain EC may be a promising therapeutic strategy for the treatment of GBM.

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.

Feeding lactate for CHO cell culture processes: Impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.