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Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs. The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

Cellular uptake of vitamin B

Designing effective therapies with selective toxicity in cancer cells is a priority of cancer research. Porphyrins are known to selectively accumulate in cancerous tissue, a feature that has been exploited in cancer diagnostics and therapeutics. The porphyrin TCPP is used in the CyPath® Lung test for diagnosis of lung cancer. During efforts to elucidate the mechanism by which TCPP selectively accumulates in cancer cells, we discovered that simultaneous knockdown of two LDLR-family receptors, CD320 and LRP2, was selectively cytotoxic to cancer cells and not to normal cells. CD320 is a receptor that internalizes the complex of cobalamin (Vitamin B12)/transcobalamin II. LRP2 is a receptor strongly expressed in the kidney which also internalizes the cobalamin/transcobalamin II complex, in addition to more than 50 other ligands. Cobalamin is the catalytic cofactor for the enzymes methionine synthase and methylmalonyl CoA mutase, which play direct and indirect roles in folate metabolism, nucleotide biosynthesis, DNA methylation, and metabolism of fatty acids and amino acids, which suggests that cobalamin deprivation might be particularly deleterious to metabolically active cancer cells. We designed siRNAs to effectively silence CD320 and LRP2 protein expression and transfected these siRNAs into normal and cancer cell lines using commercial transfection reagents. Effective gene knockdown (up to 90%) was confirmed by western blotting. Simultaneous siRNA knockdown of CD320 and LRP2 in normal fibroblasts did not affect their cell proliferation, as measured by the Cell Titer-GLO assay. However, knockdown of CD320 and LRP2 in cancer cell lines derived from diverse tissues (lung, breast, prostate, brain, and skin cancers) inhibited cell proliferation or killed the cells by up to 80%. Interestingly, in some cell lines, when either CD320 or LRP2 were silenced individually, a concurrent increase in protein expression of the other receptor was observed, suggesting that CD320 and LRP2 compensate for each other's function; hence, silencing both receptors is required for optimal cell killing. Ongoing studies will test the hypothesis that cobalamin deficiency is a mechanism for the selective cytotoxicity observed in cancer cells. Further studies include in vivo animal studies to study the effects on CD320 and LRP2 knockdown on human tumor xenograft models. In summary, we have found that inhibiting CD320 and LRP2 expression strongly and selectively inhibits cancer cell growth, leading to a promising therapeutic strategy for diverse cancers. Citation Format: David J. Elzi, William Bauta, Shao-Chiang Lai, Trisha Das, Shweta Mogare, Vivienne I. Rebel. Simultaneous knockdown of CD320 and LRP2 receptors is selectively toxic to cancer cells but not normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1223.

Cellular uptake of vitamin B12 (cobalamin, Cbl) is mediated by a cell surface receptor (TCblR/CD320) that binds transcobalamin (TC) saturated with Cbl. TC is secreted by the vascular endothelium, has a relatively short half-life, binds Cbl with high affinity and presents the vitamin to the receptor for cellular uptake. Here we show binding and internalization of the TC-Cbl complex along with its’ receptor (TCblR) in several human cell lines. The expression of TCblR is linked to the cell cycle with highest expression in actively proliferating cells. Upon binding TC-Cbl, the receptors appear to segregate on the plasma membrane and are internalized over the course of 30–60 min. Subsequently, the receptors appear to be destroyed along with the TC, which results in the release of free Cbl in the lysosome. The appearance of TCblR on the cell surface is limited to newly synthesized protein without contribution from recycling of the receptor. Therefore, Cbl uptake into cells is fully dependent on the expression of newly synthesized TCblR that is up-regulated in actively proliferating cells. The cell cycle-associated up-regulation of TCblR in cancers provides a route for targeted drug delivery.

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is a transcription factor of fundamental importance in driving lung maturation and morphogenesis. In the last decade, scientists began to appreciate the functional roles of TTF-1 in lung tumorigenesis. This movement was triggered by the discoveries of genetic alterations of TTF-1 in the form of gene amplification in lung cancer. Many downstream target genes of TTF-1 relevant to the lung cancer biology of TTF-1 have been documented. One of the most surprising findings was that TTF-1 may exhibit either pro- or antitumorigenic activities, an outcome with the complexity exceeding the original anticipation purely based on the fact that TTF-1 undergoes gene amplification in lung cancer. In the coming decade, we believe, we will witness additional surprises as the research exploring the cancer roles of TTF-1 progresses.

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is a transcription factor of fundamental importance in driving lung maturation and morphogenesis. In the last decade, scientists began to appreciate the functional roles of TTF-1 in lung tumorigenesis. This movement was triggered by the discoveries of genetic alterations of TTF-1 in the form of gene amplification in lung cancer. Many downstream target genes of TTF-1 relevant to the lung cancer biology of TTF-1 have been documented. One of the most surprising findings was that TTF-1 may exhibit either pro- or antitumorigenic activities, an outcome with the complexity exceeding the original anticipation purely based on the fact that TTF-1 undergoes gene amplification in lung cancer. In the coming decade, we believe, we will witness additional surprises as the research exploring the cancer roles of TTF-1 progresses.

Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1+ lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1+ lung cancer cells (designated EDM-TTF-1+) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1+ conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1+ lung adenocarcinomas.

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC-Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.

Cellular uptake of cobalamin (Cbl) is mediated by the transcobalamin receptor (TCblR) that binds and internalizes transcobalamin (TC) saturated with Cbl. These receptors are expressed in actively proliferating cells and are down-regulated in quiescent cells. The 5’ region of TCblR gene was analyzed for promoter activity to determine transcriptional regulation of TCblR expression. The region −668 to −455 appears to regulate TCblR expression. We have identified transcription factors MZF-1 (myeloid zinc finger 1)/RREB-1 (Ras-responsive element binding protein 1), C/EBP (CCAAT/enhancer binding protein)/HNF-3β (hepatocyte nuclear factor 3) and AP-1(activator protein 1) as proteins likely to be involved in this regulation with the former region primarily involved in up regulation and the latter two regions involved in suppression of TCblR expression. These transcription factors are involved in cell proliferation and differentiation. Thus the cell cycle associated expression of TCblR appears to be tightly regulated in synchrony with the proliferative phase of the cell cycle.

The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P

In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3′-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3′-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.

Background DNA methylation is an epigenetic phenomenon that can modulate gene function by up or downregulation of gene expression. Vitamin B12 and folate pathways are involved in the production of S-Adenosylmethionine, the universal methyl donor. Findings Brain vitamin B12 concentration and global DNA methylation was determined in transcobalamin receptor (TCblR/CD320) knock out (KO) (n = 4) and control mice (n = 4) at 20–24 weeks of age. Median [IQR] brain vitamin B12 concentrations (pg/mg) in TCblR/CD320 KO mice compared with control mice was 8.59 [0.52] vs 112.42 [33.12]; p CD320 KO compared with control mice (Median [IQR]: 0.31[0.16] % vs 0.55[0.15] %; p Conclusions In TCblR/CD320 KO mice, brain vitamin B12 drops precipitously by as much as 90% during a 20 week period. This decrease is associated with a 40% decrease in global DNA methylation in the brain. Future research will reveal whether the disruption in gene expression profiles due to changes in DNA hypomethylation contribute to central nervous system pathologies that are frequently seen in vitamin B12 deficiency.

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function.