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Abstract Circulating Tumor Cells (CTCs) in blood encompass DNA, RNA, and protein biomarkers, but clinical utility is limited by their rarity. To enable tumor epitope-agnostic interrogation of large blood volumes, we developed a high-throughput microfluidic device, depleting hematopoietic cells through high-flow channels and force-amplifying magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from seven patients with metastatic cancer. High CTC yields (mean 10,057 CTCs per patient; range 100 to 58,125) reveal considerable intra-patient heterogeneity. CTC size varies within patients, with 67% overlapping in diameter with WBCs. Paired single-cell DNA and RNA sequencing identifies subclonal patterns of aneuploidy and distinct signaling pathways within CTCs. In prostate cancers, a subpopulation of small aneuploid cells lacking epithelial markers is enriched for neuroendocrine signatures. Pooling of CNV-confirmed CTCs enables whole exome sequencing with high mutant allele fractions. High-throughput CTC enrichment thus enables cell-based liquid biopsy for comprehensive monitoring of cancer.

Abstract Immune checkpoint blockade (ICB) has demonstrated efficacy in patients with melanoma, but many exhibit poor responses. Using single cell RNA sequencing of melanoma patient-derived circulating tumor cells (CTCs) and functional characterization using mouse melanoma models, we show that the KEAP1/NRF2 pathway modulates sensitivity to ICB, independently of tumorigenesis. The NRF2 negative regulator, KEAP1, shows intrinsic variation in expression, leading to tumor heterogeneity and subclonal resistance.

Summary: TGF-β induces senescence in embryonic tissues. Whether TGF-β in the hypoxic tumor microenvironment (TME) induces senescence in cancer and how the ensuing senescence-associated secretory phenotype (SASP) remodels the cellular TME to influence immune checkpoint inhibitor (ICI) responses are unknown. We show that TGF-β induces a deeper senescent state under hypoxia than under normoxia; deep senescence correlates with the degree of E2F suppression and is marked by multinucleation, reduced reentry into proliferation, and a distinct 14-gene SASP. Suppressing TGF-β signaling in tumors in an immunocompetent mouse lung cancer model abrogates endogenous senescent cells and suppresses the 14-gene SASP and immune infiltration. Untreated human lung cancers with a high 14-gene SASP display immunosuppressive immune infiltration. In a lung cancer clinical trial of ICIs, elevated 14-gene SASP is associated with increased senescence, TGF-β and hypoxia signaling, and poor progression-free survival. Thus, TME-induced senescence may represent a naturally occurring state in cancer, contributing to an immune-suppressive phenotype associated with immune therapy resistance.

Summary: Circulating tumor cells (CTCs) enter the vasculature from solid tumors and disseminate widely to initiate metastases. Mining the metastatic-enriched molecular signatures of CTCs before, during, and after treatment holds unique potential in personalized oncology. Their extreme rarity, however, requires isolation from large blood volumes at high yield and purity, yet they overlap leukocytes in size and other biophysical properties. Additionally, many CTCs lack EpCAM that underlies much of affinity-based capture, complicating their separation from blood. Here, we provide a comprehensive introduction of CTC isolation technology, by analyzing key separation modes and integrated isolation strategies. Attention is focused on recent progress in microfluidics, where an accelerating evolution is occurring in high-throughput sorting of cells along multiple dimensions.

The mechanisms underlying the growth of breast cancer metastasis in the brain are unclear. Here, the authors use an intracranial injection mouse model and single-cell analysis of patient circulating tumour cells to demonstrate that increased hypoxic and HIF1A signalling promotes tumour growth in the brain.

Metastatic dissemination contributes to the lethality in pancreatic ductal adenocarcinoma (PDAC). Here, the authors perform RNA-sequencing on patient derived circulating tumor cells (CTCs) and identify three major CTC subgroups, and show the therapeutic potential of targeting LIN28B/let-7 pathway to halt cancer metastasis.

Abstract Background Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. Results We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. Conclusions These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.

SETD1A, a histone H3K4 methyltransferase that promotes gene expression, is required for embryonic development. Here, the authors show that SETD1A regulates the expression of mitotic genes and that SETD1A suppression induces senescence.

Abstract Background Microfluidic platforms have demonstrated the ability to isolate rare circulating tumor cells from a wide variety of cancers. Our group has recently shown the ability to isolate circulating tumor cells (CTCs) from hepatocellular carcinoma (HCC) patients using a hematopoietic cell depletion microfluidic platform. However, the relationship of CTC generation and HCC progression is still not well understood. Tumor size is often used as a clinical prognostic factor, but there has been an inconsistent relationship of tumor size and metastatic recurrence. Characterizing the relationship of primary tumor size and CTCs would provide a better understanding of HCC tumor size and metastatic potential. Results CTCs in a cohort of HCC patients with quantitative tumor volume analysis was performed to determine if there was a relationship of tumor size to CTC generation. A total of 24 tumor volumetric analyses were used in this study, and a cutoff of 25 cc was used to create a low and high tumor volume group (median 5.56 vs 108 cc; p

The current FDA-approved whole blood stabilization method for circulating tumor cell (CTC) isolation suffers from RNA degradation. Here the authors combine hypothermic preservation and antiplatelet strategies to stabilize whole blood up to 72 h without compromising CTC yield and RNA integrity.

Abstract Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm–50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15–20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

Abstract The deterioration of whole blood ex vivo represents a logistical hurdle in clinical and research settings. Here, a cocktail preservative is described that stabilizes leukocyte viability and erythrocyte morphology in whole blood under ambient storage. Neutrophil biostabilization was explored using a sophisticated microfluidic assay to examine the effectiveness of caspase inhibition to stabilize purified neutrophils. Following 72 h ambient storage, neutrophils remained fully functional to migrate towards chemical cues and maintained their ability to undergo NETosis after stimulation. Furthermore, stored neutrophils exhibited improved CD45 biomarker retention and reduced apoptosis and mortality compared to untreated controls. To stabilize erythrocyte morphology, a preservative solution was formulated using Taguchi methods of experimental design, and combined with the caspase inhibitor to form a whole blood cocktail solution, CSWB. CSWB was evaluated in blood from healthy donors and from women with metastatic breast cancer stored under ambient conditions for 72 h. CSWB-treated samples showed a significant improvement in erythrocyte morphology compared to untreated controls. Leukocytes in CSWB-treated blood exhibited significantly higher viability and CD45 biomarker retention compared to untreated controls. This 72 h shelf life under ambient conditions represents an opportunity to transport isolates or simply ease experimental timelines where blood degradation is problematic.

Abstract Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

Circulating tumour cells contribute to metastatic spread. Here the authors find that beta-chain of haemoglobin is overexpressed in those cells and protects them from oxidative stress, prolonging their survival in circulation and thereby increasing the likelihood of metastasis formation.

Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice and rapidly declined after B-RAF inhibitor treatment. CTCs were shed early from localized tumors, and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled a comparison of RNA-sequencing profiles with matched primary tumors. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF-targeted therapy. Together, the capture and molecular characterization of CTCs provide insight into the hematogenous spread of melanoma.

Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6–48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.

Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, ultimately decreasing the emergence of acquired resistance, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to its induction of SOX2, which in turn represses the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death associated with the withdrawal of growth factor signaling and may therefore contribute to the development of resistance.