Whole-genome sequencing has made a significant impact on cancer research, but traditional bulk methods fail to detect information from rare cells. Recently developed single-cell sequencing methods have provided new insights and unprecedented details about cancer progression and diversity. These advancements also enable the investigation of rare cells, such as circulating tumor cells (CTCs) derived from cancer patients. In this review, we outline various single-cell sequencing techniques that can elucidate the molecular properties of CTCs. In addition, we explain the drawbacks that need to be overcome for each method.

The PI3K/AKT/mTOR axis promotes survival and is a frequently mutated pathway in cancer. Yet, inhibitors targeting this pathway are insufficient to induce cancer cell death as single agents in some contexts, including diffuse large B cell lymphoma (DLBCL). In these situations, combinations with inhibitors targeting BCL-2 survival proteins (ABT-199 and ABT-263) may hold potential. Indeed, studies have demonstrated marked synergy in contexts where PI3K/mTOR inhibitors suppress expression of the pro-survival protein, MCL-1. In this study, we use BH3 profiling to confirm that BCL-2 and BCL-XL support survival following PI3K pathway inhibition, and that the dual PI3K/mTOR inhibitor BEZ235 strongly synergizes with BCL-2 antagonists in DLBCL. However, we identify an alternative mechanism of synergy between PI3K/mTOR and BCL-2 inhibitors, independent of MCL-1 down-regulation. Instead, we show that suppression of AKT activation by BEZ235 can induce the mitochondrial accumulation of pro-apoptotic BAD and BIM, and that expression of a constitutively active form of AKT prevents sensitization to BCL-2 antagonism. Thus, our work identifies an additional mechanism of synergy between PI3K pathway inhibitors and BCL-2 antagonists that strengthens the rationale for testing this combination in DLBCL.

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from the crew at different stages of the mission, including before (L-92, L-44, L-3 days), during (FD1, FD2, FD3), and after (R+1, R+45, R+82, R+194 days) spaceflight, creating a longitudinal sample set. The collection process included samples such as venous blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies, which were processed to obtain aliquots of serum, plasma, extracellular vesicles, and peripheral blood mononuclear cells. All samples were then processed in clinical and research laboratories for optimal isolation and testing of DNA, RNA, proteins, metabolites, and other biomolecules. This paper describes the complete set of collected biospecimens, their processing steps, and long-term biobanking methods, which enable future molecular assays and testing. As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can also aid future experiments in human spaceflight and space biology.

The PI3K/AKT/mTOR axis is one of the most commonly mutated pathways in cancer, where aberrant activation promotes cell growth, proliferation, and survival. However, selective inhibitors targeting PI3K/AKT/mTOR have been hindered by their inability to effectively induce cell death in certain cancers. With the introduction of inhibitors that directly inhibit key pro-survival factors such as BCL-2 and BCL-XL (ABT-263 and ABT-199), the potential to achieve cancer cell death using combinations of targeted inhibitors has become a realizable goal. Here we show that selective inhibition of key components in the PI3K pathway (PI3K, AKT, or mTOR) potently sensitized a panel of DLBCL cell lines to ABT-263-induced apoptosis. While the degree of sensitization varied according to which PI3K pathway component was targeted, dual inhibition of both PI3K and mTOR consistently elicited the most potent sensitization across several cell lines. Previous work in other cancer types has linked the potency of this combination to the capacity of PI3K pathway inhibitors to reduce MCL-1 in an mTORC1-dependent manner. However, we found that this was not the case for DLBCL. For example, ABT-263 resistance induced by over-expression of MCL-1 was overcome when cells were co-treated with a dual-PI3K/mTOR inhibitor NVP-BEZ235 despite maintained expression of MCL-1. Instead, inhibition of the PI3K pathway led to a general increase in mitochondrial priming as measured by BH3 profiling. This occurred through distinct effects from both AKT and mTORC1 on the abundance of multiple BCL-2 family proteins at the mitochondria including reductions in the pro-survival proteins BCL-2, MCL-1, and BCL-XL as well as increases in Bim and Bad. In addition to these direct effects on BCL-2 family proteins, we found that inhibition of the PI3K pathway reduced expression of cancer-relevant proteins c-Myc and eIF4G as determined by reverse phase protein array. Thus, this project highlights the broad effects of dual-PI3K/mTOR inhibitors that sensitize DLBCL to apoptosis, and suggests that inhibition of the PI3K pathway has distinct effects on cell survival signaling among different cancer cell types. Citation Format: Jong-Hoon Scott Lee, Sarah Tang, Veronica Ortiz, David A. Fruman. Vertical inhibition of the PI3K pathway potently sensitizes diffuse large B cell lymphoma to BCL-2 antagonism. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B45.

The I4 mission, the first all-civilian orbital flight mission, investigated the physiological effects of short duration spaceflight through a multi-omic approach. We analyzed telomere length, clonal hematopoiesis of indeterminate potential (CHIP), whole genome stability, cell-free DNA (cfDNA) cell lysis, and immune cell gene expression. Our results revealed telomere length dynamics similar to those observed in the NASA Twins Study and in astronauts spending 6 months on the ISS, with lengthening in space and shortening upon return to Earth. Our cell-type of origin analysis of cfDNA fragments revealed an increased presence of innate and adaptive immune cell signatures that persisted over a month after return to earth. No significant relationship between spaceflight and CHIP-related or whole genome abnormalities were observed. Longitudinal mitochondrial, ribosomal and immune function gene expression changes occurred across both adaptive and innate immune cells, suggesting adaptations to the space environment can extend months after return from spaceflight and alter immune function. Our findings provide valuable insights into the physiological consequences of short duration spaceflight and will serve as a reference point for future space tourism, low Earth-orbit (LEO) missions, and deep-space exploration.

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from the crew at different stages of the mission, including before (L-92, L-44, L-3 days), during (FD1, FD2, FD3), and after (R + 1, R + 45, R + 82, R + 194 days) spaceflight, creating a longitudinal sample set. The collection process included samples such as venous blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies, which were processed to obtain aliquots of serum, plasma, extracellular vesicles, and peripheral blood mononuclear cells. All samples were then processed in clinical and research laboratories for optimal isolation and testing of DNA, RNA, proteins, metabolites, and other biomolecules. This paper describes the complete set of collected biospecimens, their processing steps, and long-term biobanking methods, which enable future molecular assays and testing. As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can also aid future experiments in human spaceflight and space biology.

SummaryHypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis and has been associated with tumor progression and metastasis. Most of the well- known hypoxia effects are mediated through hypoxia-inducible factors (HIFs), but the long- lasting effect of hypoxia beyond the immediate HIF regulation remains less understood. Here we show that hypoxia exerts a prolonged effect through HIF-independent mechanisms to promote metastasis. Using breast cancer patient-derived circulating tumor cell (CTC) lines and common breast cancer cell lines, we found that hypoxia downregulates tumor intrinsic type I interferon (IFN) signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells. This effect does not depend on the canonical HIFs but is mediated, at least partially, via forkhead box protein O1 (FOXO1). Moreover, hypoxia induced IFN/AP suppression can last longer than the hypoxic exposure, presenting a “hypoxic memory” phenotype. Hypoxic memory of IFN/AP downregulation is established by specific hypoxic priming, and cells with hypoxic memory have an enhanced ability for tumorigenesis and metastasis. The histone deacetylase inhibitor (HDACi) Entinostat can erase the hypoxic memory and improve the immune clearance of tumor cells when combined with checkpoint immunotherapies in a syngeneic breast cancer mouse model. These results point to a novel mechanism for hypoxia facilitated tumor progression, through a long-lasting memory that provides advantages for CTCs during the metastatic cascade.