A prime/boost vaccine platform efficiently identifies CD27 agonism and depletion of myeloid-derived suppressor cells as therapies that rationally combine with checkpoint blockade in ovarian cancer.

Cancer immunotherapies have generated remarkable clinical responses for some patients with advanced/metastatic disease, prompting exploration of rational combination therapies to bolster anti-tumor immunity in patients with limited response or those who experience tumor progression following an initial response to immunotherapy. In contrast to other tumor indications, objective response rates to single-agent PD-1/PD-L1 blockade in ovarian cancer are limited, suggesting a need to identify combinatorial approaches that lead to tumor regression in a setting where checkpoint blockade alone is ineffective. Using a pre-clinical model of aggressive intraperitoneal ovarian cancer, we have previously reported on a heterologous prime/boost cancer vaccine that elicits robust anti-tumor immunity, prolongs survival of tumor-bearing mice, and which is further improved when combined with checkpoint blockade. As tumor control in this model is CD8 + T cell dependent, we reasoned that the prime/boost vaccine platform could be used to explore additional treatment combinations intended to bolster the effects of CD8 + T cells. Using whole tumor transcriptomic data, we identified candidate therapeutic targets anticipated to rationally combine with prime/boost vaccination. In the context of a highly effective cancer vaccine, CD27 agonism or antibody-mediated depletion of granulocytic cells each modestly increased tumor control following vaccination, with anti-PD-1 therapy further improving treatment efficacy. These findings support the use of immunotherapies with well-defined mechanisms(s) of action as a valuable platform for identifying candidate combination approaches for further therapeutic testing in ovarian cancer.

Conserved features of selenocysteine insertion sequence (SECIS) elements in selenoprotein W cDNAs from five species.

SECIS elements form stem-loop structures in the 3' untranslated regions (UTR) of eukaryotic mRNAs that encode selenoproteins. These elements direct incorporation of selenocysteine at UGA codons, provided the SECIS element lies a sufficient distance from the UGA. The cDNAs encoding skeletal muscle selenoprotein W from human, rhesus monkey, sheep, rat, and mouse contained highly similar SECIS elements that retained important features common to all known SECIS elements. Comparative analysis of these SECIS elements showed that in some regions both predicted secondary structure and nucleotide sequences were conserved, in other areas secondary structure was maintained using different primary sequence, and in still other portions, base pairing was not conserved. The rodent and sheep selenoprotein W mRNAs used UGA as a stop codon and as a selenocysteine codon. Thus, UGA specified both selenocysteine incorporation and termination in a single mRNA. The selenoprotein W SECIS elements contained an additional highly conserved base-paired stem that may prevent inappropriate selenocysteine incorporation at the UGA stop codons.

Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.