LDH-A-Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses.

Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors.

Genetic and Drug Inhibition of LDH-A: Effects on Murine Gliomas.

The effects of the LDH-A depletion via shRNA knockdown on three murine glioma cell lines and corresponding intracranial (i.c.) tumors were studied and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of genetic-shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar. LDH-A KD and GNE-R-140 unexpectedly increased the aggressiveness of GL261 intracranial gliomas, but not CT2A and ALTS1C1 i.c. gliomas. Furthermore, the bioenergetic profiles (ECAR and OCR) of GL261 NC and LDH-A KD cells under different nutrient limitations showed that (a) exogenous pyruvate is not a major carbon source for metabolism through the TCA cycle of native GL261 cells; and (b) the unique upregulation of LDH-B that occurs in GL261 LDH-A KD cells results in these cells being better able to: (i) metabolize lactate as a primary carbon source through the TCA cycle, (ii) be a net consumer of lactate, and (iii) showed a significant increase in the proliferation rate following the addition of 10 mM lactate to the glucose-free media (only seen in GL261 KD cells). Our study suggests that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, since the level of LDH-A expression and its interplay with LDH-B can lead to complex metabolic interactions between tumor cells and their environment. Metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis (e.g., inhibition of LDH-A) may lead to the unexpected development and activation of alternative metabolic pathways (e.g., upregulation of lipid metabolism and fatty-acid oxidation pathways), resulting in enhanced tumor-cell survival in a nutrient-limited environment and leading to increased tumor aggressiveness.

CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8+ T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (Treg) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of Treg cells is dependent on Treg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with Treg cell function in the presence of glucose.

Lactate Dehydrogenase A Depletion Alters MyC-CaP Tumor Metabolism, Microenvironment, and CAR T Cell Therapy.

To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA+ MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA+ Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors. However, total tumor lactate concentration did not differ significantly between LDH-A knockdown and control tumors, reflecting the lower vascularity, blood flow, and clearance of lactate from LDH-A knockdown tumors. Comparing treatment responses of MyC-CaP tumors with LDH-A depletion and/or anti-hPSMA CAR T cells showed that the dominant effect on tumor growth was LDH-A depletion. With anti-hPSMA CAR T cell treatment, tumor growth was significantly slower when combined with tumor LDH-A depletion and compared to control tumor growth (p < 0.0001). The lack of a complete tumor response in our animal model can be explained in part by (1) the lower activity of human CAR T cells against hPSMA-expressing murine tumors in a murine host, and (2) a loss of hPSMA antigen from the tumor cell surface in progressive generations of tumor cells.

Imaging CXCR4 Expression with Iodinated and Brominated Cyclam Derivatives.

PURPOSE: CXCR4 is one of several "chemokine" receptors expressed on malignant tumors (including GBM and PCNSL) and hematopoietic stem cells. Although 68Ga-pentixafor and 68Ga-NOTA-NFB have been shown to effectively image CXCR4 expression in myeloma and other systemic malignancies, imaging CXCR4 expression in brain tumors has been more limited due to the blood-brain barrier (BBB) and a considerable fraction of CXCR4 staining is intracellular. METHODS: We synthesized 6 iodinated and brominated cyclam derivatives with high affinity (low nM range) for CXCR4, since structure-based estimates of lipophilicity suggested rapid transfer across the BBB and tumor cell membranes. RESULTS: We tested 3 iodinated and 3 brominated cyclam derivatives in several CXCR4(+) and CXCR4(-) cell lines, with and without cold ligand blocking. To validate these novel radiolabeled cyclam derivatives for diagnostic CXCR4 imaging efficacy in brain tumors, we established appropriated murine models of intracranial GBM and PCNSL. Based on initial studies, 131I-HZ262 and 76Br-HZ270-1 were shown to be the most avidly accumulated radioligands. 76Br-HZ270-1 was selected for further study in the U87-CXCR4 and PCNSL #15 intracranial tumor models, because of its high uptake (9.5 ± 1.3 %ID/g, SD) and low non-specific uptake (1.6 ± 0.7 %ID/g, SD) in the s.c. U87-CXCR4 tumor models. However, imaging CXCR4 expression in intracranial U87-CXCR4 and PCNSL #15 tumors with 76Br-HZ270-1 was unsuccessful, following either i.v. or spinal-CSF injection. CONCLUSIONS: Imaging CXCR4 expression with halogenated cyclam derivatives was successful in s.c. located tumors, but not in CNS located tumors. This was largely due to the following: (i) the hydrophilicity of the radiolabeled analogues-as reflected in the "measured" radiotracer distribution (LogD) in octanol/PBS-which stands in contrast to the structure-based estimate of LogP, which was the rationale for initiating the study and (ii) the presence of a modest BTB in intracranial U87-CXCR4 gliomas and an intact BBB/BTB in the intracranial PCNSL animal model.

LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response.

Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.

Synthesis and evaluation of an (18)F-labeled pyrimidine-pyridine amine for targeting CXCR4 receptors in gliomas.

INTRODUCTION: Chemokine receptor-4 (CXCR4, fusin, CD184) is expressed on several tissues involved in immune regulation and is upregulated in many diseases including malignant gliomas. A radiolabeled small molecule that readily crosses the blood-brain barrier can aid in identifying CXCR4-expressing gliomas and monitoring CXCR4-targeted therapy. In the current work, we have synthesized and evaluated an [(18)F]-labeled small molecule based on a pyrimidine-pyridine amine for its ability to target CXCR4. EXPERIMENTAL: The nonradioactive standards and the nitro precursor used in this study were prepared using established methods. An HPLC method was developed to separate the nitro-precursor from the nonradioactive standard and radioactive product. The nitro-precursor was radiolabeled with (18)F under inert, anhydrous conditions using the [(18)F]-kryptofix 2.2.2 complex to form the desired N-(4-(((6-[(18)F]fluoropyridin-2-yl)amino)methyl)benzyl)pyrimidin-2-amine ([(18)F]-3). The purified radiolabeled compound was used in serum stability, partition coefficient, cellular uptake, and in vivo cancer targeting studies. RESULTS: [(18)F]-3 was synthesized in 4-10% decay-corrected yield (to start of synthesis). [(18)F]-3 (tR ≈ 27 min) was separated from the precursor (tR ≈ 30 min) using a pentafluorophenyl column with an isocratic solvent system. [(18)F]-3 displayed acceptable serum stability over 2 h. The amount of [(18)F]-3 bound to the plasma proteins was determined to be > 97%. The partition coefficient (LogD7.4) is 1.4 ± 0.5. Competitive in vitro inhibition indicated 3 does not inhibit uptake of (67)Ga-pentixafor. Cell culture media incubation and ex vivo urine analysis indicate rapid metabolism of [(18)F]-3 into hydrophilic metabolites. Thus, in vitro uptake of [(18)F]-3 in CXCR4 overexpressing U87 cells (U87 CXCR4) and U87 WT indicated no specific binding. In vivo studies in mice bearing U87 CXCR4 and U87 WT tumors on the left and right shoulders were carried out using [(18)F]-3 and (68)Ga-pentixafor on consecutive days. The CXCR4 positive tumor was clearly visualized in the PET study using (68)Ga-pentixafor, but not with [(18)F]-3. CONCLUSIONS: We have successfully synthesized both a radiolabeled analog to previously reported CXCR4-targeting molecules and a nitro precursor. Our in vitro and in vivo studies indicate that [(18)F]-3 is rapidly metabolized and, therefore, does not target CXCR4-expressing tumors. Optimization of the structure to improve the in vivo (and in vitro) stability, binding, and solubility could lead to an appropriate CXCR4-targeted radiodiagnositic molecule.

[Cis-diamminedichloroplatinum penetration into the cerebrospinal fluid of the lateral ventricle, postoperative cavity, and lumbar subarachnoid space with or without pre-intravenous mannitol administration in patients with brain metastasis from lung cancer].

We have previously reported that repeated intravenous administration of cis-diamminedichloroplatinum(CDDP)prevented local recurrence of metastatic brain tumors after surgical total excision; however, data on CDDP distribution in the postoperative cavity after intravenous CDDP administration are not available. In the present study, we evaluated the penetration of total platinum(Pt)into the cerebrospinal fluid(CSF)and the effect of intravenous administration of 20%mannitolon total Pt distribution in the plasma and CSF. Total Pt levels in the plasma and CSF were determined immediately after intravenous infusion of CDDP(80mg/m2)for 1 hour, with or without pre-intravenous infusion of 20% mannitol(200mL), in 11 patients with brain metastasis from lung cancer. CSF samples were obtained via Ommaya reservoirs placed in the anterior horn of the lateral ventricle(CSF-V)and the postoperative cavity(CSF-C). Spinal CSF (CSF-L)was also obtained in the last 4 patients of the series via spinal drainage. CDDP was administered intravenously without mannitol 10 days after brain tumor excision, and 1 week after the initial administration of CDDP, CDDP was intravenously administered again after intravenous mannitol administration. CDDP was always administered intravenously at 1:00 PM to rule out the influence of circadian variation. Plasma and CSF were sequentially sampled after intravenous CDDP administration, and their Pt levels were analyzed for Pt content by using atomic absorption spectroscopy. The area under the concentration time curve(AUC)was calculated for plasma and CSF using the moment method. The AUC for total Pt in plasma showed a significantly higher level with the administration of mannitol than without the administration of mannitol(p<0.01, paired t-test). Total Pt levels(AUCs and peak concentrations)in CSF-C were much higher than were those in CSF-V and CSF-L both with and without the administration of mannitol. The ratio(%)of CDDP penetration into CSF(CSF AUC/plasma AUC)was much higher for CSF-C than for CSF-V(p<0.0001)both with and without mannitol administration. However, the CSF penetration ratio with mannitol administration did not differ significantly from that without mannitol administration. Thus, the administration of mannitol did not significantly increase the penetration ratio of total Pt. Creatinine clearance was moderately reduced in all patients during the first administration of CDDP. However, 7 of the 11 patients showed an increase in creatinine clearance after the second administration of CDDP with mannitol.