Fatty acid oxidation fuels natural killer cell responses against infection and cancer.

Natural killer (NK) cells are a vital part of the innate immune system capable of rapidly clearing mutated or infected cells from the body and promoting an immune response. Here, we find that NK cells activated by viral infection or tumor challenge increase uptake of fatty acids and their expression of carnitine palmitoyltransferase I (CPT1A), a critical enzyme for long-chain fatty acid oxidation. Using a mouse model with an NK cell-specific deletion of CPT1A, combined with stable 13C isotope tracing, we observe reduced mitochondrial function and fatty acid-derived aspartate production in CPT1A-deficient NK cells. Furthermore, CPT1A-deficient NK cells show reduced proliferation after viral infection and diminished protection against cancer due to impaired actin cytoskeleton rearrangement. Together, our findings highlight that fatty acid oxidation promotes NK cell metabolic resilience, processes that can be optimized in NK cell-based immunotherapies.

Preclinical Development and Characterization of Novel Adeno-Associated Viral Vectors for the Treatment of Lipoprotein Lipase Deficiency.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing, and vector-related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-Good Laboratory Practice toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon-optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared with an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

The Ratio of Key Metabolic Transcripts Is a Predictive Biomarker of Breast Cancer Metastasis to the Lung.

Understanding the rewired metabolism underlying organ-specific metastasis in breast cancer could help identify strategies to improve the treatment and prevention of metastatic disease. Here, we used a systems biology approach to compare metabolic fluxes used by parental breast cancer cells and their brain- and lung-homing derivatives. Divergent lineages had distinct, heritable metabolic fluxes. Lung-homing cells maintained high glycolytic flux despite low levels of glycolytic intermediates, constitutively activating a pathway sink into lactate. This strong Warburg effect was associated with a high ratio of lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) expression, which correlated with lung metastasis in patients with breast cancer. Although feature classification models trained on clinical characteristics alone were unable to predict tropism, the LDH/PDH ratio was a significant predictor of metastasis to the lung but not to other organs, independent of other transcriptomic signatures. High lactate efflux was also a trait in lung-homing metastatic pancreatic cancer cells, suggesting that lactate production may be a convergent phenotype in lung metastasis. Together, these analyses highlight the essential role that metabolism plays in organ-specific cancer metastasis and identify a putative biomarker for predicting lung metastasis in patients with breast cancer. SIGNIFICANCE: Lung-homing metastatic breast cancer cells express an elevated ratio of lactate dehydrogenase to pyruvate dehydrogenase, indicating that ratios of specific metabolic gene transcripts have potential as metabolic biomarkers for predicting organ-specific metastasis.

Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

A low carbohydrate, high protein diet slows tumor growth and prevents cancer initiation.

Since cancer cells depend on glucose more than normal cells, we compared the effects of low carbohydrate (CHO) diets to a Western diet on the growth rate of tumors in mice. To avoid caloric restriction-induced effects, we designed the low CHO diets isocaloric with the Western diet by increasing protein rather than fat levels because of the reported tumor-promoting effects of high fat and the immune-stimulating effects of high protein. We found that both murine and human carcinomas grew slower in mice on diets containing low amylose CHO and high protein compared with a Western diet characterized by relatively high CHO and low protein. There was no weight difference between the tumor-bearing mice on the low CHO or Western diets. Additionally, the low CHO-fed mice exhibited lower blood glucose, insulin, and lactate levels. Additive antitumor effects with the low CHO diets were observed with the mTOR inhibitor CCI-779 and especially with the COX-2 inhibitor Celebrex, a potent anti-inflammatory drug. Strikingly, in a genetically engineered mouse model of HER-2/neu-induced mammary cancer, tumor penetrance in mice on a Western diet was nearly 50% by the age of 1 year whereas no tumors were detected in mice on the low CHO diet. This difference was associated with weight gains in mice on the Western diet not observed in mice on the low CHO diet. Moreover, whereas only 1 mouse on the Western diet achieved a normal life span, due to cancer-associated deaths, more than 50% of the mice on the low CHO diet reached or exceeded the normal life span. Taken together, our findings offer a compelling preclinical illustration of the ability of a low CHO diet in not only restricting weight gain but also cancer development and progression.

Validation of cell density and viability assays using Cedex automated cell counter.

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-to-instrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13x10(5) to approximately 1.0x10(7)cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3-106.4% recovery) and precision (RSD<5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.

Mammary tumors with diverse immunological phenotypes show differing sensitivity to adoptively transferred CD8+ T cells lacking the Cbl-b gene.

We tested the efficacy of CD8+ T cells lacking the Cbl-b gene against a panel of mammary tumor lines with different intrinsic sensitivities to T cells. Mice bearing established tumors expressing an ovalbumin-tagged version of HER-2/neu underwent adoptive transfer with Cbl-b-replete or -null CD8+ T cells from OT-I T cell receptor transgenic donor mice. In general, Cbl-b-null OT-I cells showed enhanced expansion, persistence, and capacity for tumor infiltration. This resulted in markedly enhanced efficacy against two tumor lines that normally demonstrate complete (NOP21) or partial (NOP23) regression. Moreover, a third tumor line (NOP6) that normally demonstrates progressive disease underwent complete regression in response to Cbl-b-null OT-I cells. However, a fourth tumor line (NOP18) was resistant to Cbl-b-null OT-I cells owing to a profound barrier to lymphocyte infiltration. Thus, Cbl-b-null CD8+ T cells are generally more efficacious but are nonetheless unable to mediate curative responses against all tumor phenotypes.

Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo.

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been implicated in the differentiation and growth inhibition of cancer cells. We examined the effects of PPARgamma activation by troglitazone on hepatocellular carcinoma (HCC) cell growth, proliferation, and apoptosis in vitro and in vivo. We also studied relationships between PPARgamma activation and cyclooxygenase-2 (COX-2) expression. Human HCC cell lines Huh7 and Hep3B were cultured in the presence or absence of troglitazone. Cell growth was determined via WST-1 assay, proliferation by cell cycle analysis and proliferating cell nuclear antigen (PCNA) Western blotting, and apoptosis by flow cytometry and TUNEL. Tumor growth after subcutaneous implantation of Huh7 cells in nude mice was monitored, and the effects of treatment with troglitazone were determined. In resected HCCs, PPARgamma expression was less compared with the histologically normal surrounding liver. In cultures of Hep3B and Huh7 cells, basal expression of PPARgamma was relatively low, but troglitazone caused dose-dependent induction of PPARgamma expression. Cell cycle analysis revealed a decreased proportion of cells in S phase, with arrest at G0/G1. Concomitant downregulation of PCNA and an increase in TUNEL staining, cells were consistent with decreased proliferation and induction of apoptosis by troglitazaone. Troglitazone-mediated PPARgamma activation also suppressed COX-2 expression and induced p27 in HCC cells. Administration of troglitazone to Huh7 tumor-bearing mice significantly reduced tumor growth and caused tumor regression. In conclusion, collectively, these results indicate that PPARgamma could be a regulator of cell survival and growth in HCC. PPARgamma therefore represents a putative molecular target for chemopreventive therapy or inhibition of liver cancer growth.