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Non-small cell lung cancer (NSCLC) metastatic to the brain leptomeninges is rapidly fatal, cannot be biopsied, and cancer cells in the cerebrospinal fluid (CSF) are few; therefore, available tissue samples to develop effective treatments are severely limited. This study aimed to converge single-cell RNA-seq and cell-free RNA (cfRNA) analyses to both diagnose NSCLC leptomeningeal metastases (LM), and to use gene expression profiles to understand progression mechanisms of NSCLC in the brain leptomeninges. NSCLC patients with suspected LM underwent withdrawal of CSF via lumbar puncture. Four cytology-positive CSF samples underwent single-cell capture (n = 197 cells) by microfluidic chip. Using robust principal component analyses, NSCLC LM cell gene expression was compared to immune cells. Massively parallel qPCR (9216 simultaneous reactions) on human CSF cfRNA samples compared the relative gene expression of patients with NSCLC LM (n = 14) to non-tumor controls (n = 7). The NSCLC-associated gene, CEACAM6, underwent in vitro validation in NSCLC cell lines for involvement in pathologic behaviors characteristic of LM. NSCLC LM gene expression revealed by single-cell RNA-seq was also reflected in CSF cfRNA of cytology-positive patients. Tumor-associated cfRNA (e.g., CEACAM6, MUC1) was present in NSCLC LM patients’ CSF, but not in controls (CEACAM6 detection sensitivity 88.24% and specificity 100%). Cell migration in NSCLC cell lines was directly proportional to CEACAM6 expression, suggesting a role in disease progression. NSCLC-associated cfRNA is detectable in the CSF of patients with LM, and corresponds to the gene expression profile of NSCLC LM cells. CEACAM6 contributes significantly to NSCLC migration, a hallmark of LM pathophysiology.

Background Homeless street sweeps are frequent operations in many cities in the USA in which government agencies move unhoused people living in public outdoor areas. Little research exists on the health impact of street sweeps operations. Objective This study was created at the request of community advocacy groups to investigate and document the health impacts of street sweeps from the perspective of healthcare providers. Design This is a qualitative study using data gathered from open-ended questions. Participants We recruited 39 healthcare providers who provided health and wellness services in San Francisco for people experiencing homelessness (PEH) between January 2018 and January 2020. Interventions We administered a qualitative, open-ended questionnaire to healthcare providers using Qualtrics surveying their perspectives on the health impact of street sweeps. Approach We conducted qualitative thematic analysis on questionnaire results. Key Results Street sweeps may negatively impact health through two outcomes. The first outcome is material loss, including belongings and medical items. The second outcome is instability, including geographic displacement, community fragmentation, and loss to follow-up. These outcomes may contribute to less effective management of chronic health conditions, infectious diseases, and substance use disorders, and may increase physical injuries and worsen mental health. Providers also reported that sweeps may negatively impact the healthcare system by promoting increased usage of emergency departments and inpatient hospital care. Conclusions Sweeps may have several negative consequences for the physical and mental health of the PEH community and for the healthcare system.

Leptomeningeal metastases (LM), a diffuse form of brain metastases is rare and fatal progression of non-small cell lung cancer (NSCLC). In LM, metastatic cancer cells spread and resign on the brain meninges, the cerebrospinal fluid (CSF), cranial and spinal nerves. Rapid disease progression and scarce tissue availability hinder the progress of scientific study of LM and its treatment. To overcome the critical lack of tissue and to determine the genetic profile of NSCLC LM, we have developed methods to extract tumor-associated cell-free RNA from CSF, and isolated and sequenced circulating single cells from CSF. Herein, we used high throughput qPCR to target lung and brain-associated genes and identified NSCLC LM metastases-related RNA. Brain-specific gene signature (GFAP, NRGN, SNCB, ZBTB18) was detected in all CSF sample (control and metastases), whereas lung-specific genes (MUC1, SFTPB, SFTPD, SLC34A2) were detected in CSF of brain metastases patients. Normal, healthy CSF lacks cellular component, but CSF of patients with LM metastases inhabited with very low amount of circulating tumor cells. Single cells from CSF of 4 patients with NSCLC LM metastases were captured with microfluidic chip. Cells (n = 197) were clustered by significantly differential expressed genes demonstrating two distinct populations of white blood and tumor cells. These data identified specific cfRNA and single cell transcriptome profiles compared to normal cells or patients without NSCLC LM metastases, and highlighted metastases-associated carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) as highly expressed in patients with NSCLC LM metastases. CEACAM6 mRNA was detected in CSF of 86% of patients with NSCLC LM but not in the CSF of control patients. In vitro inhibition of CEACAM6 protein lead to decreased invasion in NSCLC cells which was rescued by overexpression of the protein. We have developed sensitive and robust techniques to leverage human CSF to study NSCLC LM.

Leptomeningeal metastasis (LM), a spread of cancer to the meninges and cerebrospinal fluid, carries extremely poor prognosis due to fast progression and no effective treatment. Given that LM mostly develops in specific types of cancer, notably melanoma, breast and lung cancer, it is likely that predisposition for LMD is shaped by specific genetic footprints and/or anti-cancer therapies. In this regard, it is interesting that among all breast cancers the triple-negative (i.e. estrogen and progesterone receptor-negative and HER2 overexpression-negative) breast cancer (TNBC) type develops a disproportionally high percentage of LM, accounting for the majority (~40%) of all breast cancer LM cases. TNBCs trace their origin to the genomic instability stemming from defects in DNA repair (notably homology-directed repair, HDR). We have recently shown that the efficiency of HDR depends on dynamin 2 (DNM2) best known for its role in endocytic protein trafficking. Higher DNM2 was associated with more efficient HDR and the resistance to DNA-crosslinking chemotherapy. Importantly, elevated DNM2 was associated with lower relapse-free survival and shorter times to relapse after chemotherapy only in TNBCs and not in other types of breast cancer. As DNM2 also fuels migration and invasion, the cells with high DNM2 are not only the most resistant to chemotherapy but also are the most mobile, and thus may represent the core population of LM. Here we test the inhibition of the DNM2-dependent endocytic trafficking as a potential therapeutic strategy to halt LM in TNBC. As a model of LM we use the human-in-mouse model of brain metastasis based on internal carotid injection of MDA-MB-231-BR3 cells, which we have shown to faithfully recapitulate human LM. We show that DNM2 knockdown delays metastatic spread to the brain and potentiates the effect of DNA-crosslinking chemotherapeutic cyclophosphamide, providing justification for further testing of DNM2 inhibitors for targeted therapy of LM.