B. The importance of the candidate gene LDHA in brain metastasis.

Fig. 2. LDHA expression in brain metastases. A. Different sized brain metastases in our H1_DL2 model. A decrease in LDHA level with increased tumor size is seen. B. Scoring of LDHA levels in 59 different H1_DL2 experimental brain metastases (standard Remmele pathology score, incl. staining intensity and frequency). Red=high LDHA score, blue=low LDHA score. Mean Ø of the 59 tumors was 305um, thus we compared difference in LDHA score for tumors larger than or smaller than 305um. C. Logistic regression analysis with Ø as a regressor. D. Human melanoma metastases. The highest LDHA levels are seen in hypoxic, prenecrotic areas (white arrowheads). E. The infiltration zone in an early human brain metastases from a lung adenocarcinoma, showing co-optive tumor cell growth around small vessels. LDHA positive cells were seen (black arrowheads). F. Human brain metastases from colon carcinoma, where high levels of LDHA expression was seen next to necrotic areas (white asterisks), whereas on the stromal/vessel side LDHA was almost absent (black arrowheads).

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Many cancer cells produce ATP by converting glucose to lactate, a process essential to the hypoxic environment in which many cancer cells exist. Increased serum level of lactate dehydrogenase-A (LDHA) is likely related to the hypoxic environment of tumor cells. Our RNA-seq data shows that LDHA is highly expressed at genomic (i.e. transcript) level in brain metastases in our animal model. Immunostaining of brain metastasis from animals as well as humans shows a correlation between LDHA expression levels in tumor tissue and tumor size, where LDHA is highly expressed during micrometastasis formation, and is downregulated when tumor size increases (Fig. 2). This indicates that LDHA may be crucial for establishing micrometastases in early steps of experimental brain metastasis, for example when oxygen/nutrition supply is limited due to delayed angiogenesis.

We have now stably knocked out LDHA in our H1_DL2 melanoma cell line. We are currently performing in vitro viability assays and real-time in vitro kinetic measurements of cell metabolism. We are also doing in vivo studies, by injecting LDHA knock-down cells into nod/scid mice, followed by MRI, ¹⁸F-FDG PET and ¹⁸F-FLT PET to study tumor burden and animal survival. We have access to a tissue microarray with 112 patient brain metastases, and we will correlate patient survival, radiological tumor size and number of metastases with tumor LDHA status.

18.10.2013