Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: HEPACAM2 expression in SCLC cells and tumors. (A) HEPACAM2 mRNA levels in cancer cell lines are based on RNA‐seq data from the CCLE. (B) HEPACAM2 mRNA levels were validated by qPCR and are presented as log10 of fold change relative to the β‐actin mRNA expression level (symbols show an average of three replicates per cell line). We used the RNA‐seq value for each HEPACAM2 level, log2(RPKM), downloaded from the CCLE data set found at http://cBioPortal.org , to compare our qPCR data. (C) HEPACAM2 mRNA levels in tumor samples were detected by RNA‐seq comparing SCLC ( n = 29) with other cancers in TCGA; **** P value <0.0001. (D) HEPACAM2 mRNA levels in tumor samples were detected by RNA‐seq comparing SCLC ( n = 29) to normal lung ( n = 25) and both typical ( n = 17) and atypical ( n = 13) pulmonary carcinoid tumors. BRCA indicates breast cancer; CCLE, Cancer Cell Line Encyclopedia; CR, colorectal cancer; GBM, glioblastoma; LUAD, lung adenoma; LUSC, lung squamous NSCLC; mRNA, messenger RNA; NK, natural killer; NOS, not otherwise specified; NSCLC, non–small cell lung cancer; PRAD, prostate adenoma cancer; qPCR, quantitative polymerase chain reaction; RNA‐seq, RNA sequencing; RPKM, reads per kilobase per million mapped reads; SCLC, small cell lung cancer; SKCM, skin melanoma cancer; TCGA, The Cancer Genome Atlas.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Localization of protein expression of HEPACAM2. (A) Immunofluorescence staining of HEPACAM2 in SCLC cell lines revealed that most protein expression localized to the plasma membrane. A ProSci antibody was used to detect HEPACAM2 (pseudo green) and counterstained with Hoechst (pseudo blue). (B) Immunofluorescence staining of overexpressed HEPACAM2 (pseudo green) in an NSCLC cell line, H1299, revealed that most protein expression localized to the plasma membrane (yellow arrows), as evidenced by counterstaining (pseudo pink) with a plasma membrane marker (CellBrite) ( Right ). The scale bar represents 10 µm. (C) Western blots of identical protein lysates of control and HEPACAM2‐overexpressing H1299 cells were detected with both FLAG and ProSci antibodies. Black arrowheads indicate the major ∼70‐kDa band. (D) Western blots of HEPACAM2 expression in identical protein lysates of control, vehicle‐treated and tunicamycin‐treated HEPACAM2‐overexpressing H1299 cells were detected by both FLAG and ProSci antibodies. Black arrowheads indicate the major ∼70‐kDa band; red arrowheads indicate a ∼50‐kDa band. (E) Western blots of HEPACAM2 expression in biotin pull‐down control and HEPACAM2‐overexpressing H1299 cells were detected with a ProSci antibody after streptavidin pull‐down. The black arrowhead indicates the major ∼70‐kDa band. C indicates control; Con, control; IB, immunoblotting; kDa, kilodalton; NSCLC, non–small cell lung cancer; OE, overexpressing; SCLC, small cell lung cancer; T , tunicamycin‐treated; V , vehicle‐treated.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Marker, Western Blot, Control

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: HEPACAM2 expression detected in EVs of SCLC cell lines. (A) mRNAs of HEPACAM2 could be detected in EVs of SCLC cell lines (H526, H209, and H510A) but not NSCLC cell lines (PC9, H1299, HCC15, and A427) and a normal cell line (BEAS2B). For better visualization, HEPACAM2 mRNA is shown as the ratio of log2‐transformed H2:β‐actin. An unpaired t ‐test with a Welch correction was performed with GraphPad Prism, version 10.1.0. (B) HEPACAM2 protein was also found via liquid chromatography–tandem mass spectrometry in the secretomes of NE‐specific SCLC cell lines. Results are expressed as the mean values of the data as bars, representing sample values and error bars representing the SEM. EV indicates extracellular vesicle; mRNA, messenger RNA; NE, neuroendocrine; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, Transformation Assay, Liquid Chromatography, Mass Spectrometry

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression correlates with NE carcinoma in tumors. (A) Boxplots showing mRNA levels of HEPACAM2 in NSCLC and SCLC with a two‐tailed ANOVA test. The graph displays the mean values as bars, with individual data points representing sample values and error bars indicating the SEM. (B) Spatially resolved expression of HEPACAM2 ( Top Left ) is specific to the region of SCLC histology, which expresses high NE genes (i.e., SYP and ASCL1), but not the NSCLC region, with features of adenocarcinoma (i.e., NAPSA) in a combined NSCLC/SCLC tissue sample; performed by 10x Genomics. The image was generated with the Loupe Browser, version 6.4.1. (C) Heatmap of transcription factors ordered by HEPACAM2 and z scaled by row, which allowed comparison between samples. ANOVA indicates analysis of variance; mRNA, messenger RNA; NE, neuroendocrine; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer; SEM, standard error of the mean; TPM, transcript per million.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing, Two Tailed Test, Generated, Comparison

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression and association with NE carcinoma in tumors. (A–C) In patient samples, no change in HEPACAM2 level of expression was found to be associated with different stages of disease (A), biopsy location (B), and patient response (C). (D) High levels of HEPACAM2 expression were negatively correlated with PFS and OS in our SCLC cohort. LS, limited stage; NE indicates neuroendocrine; OS, overall survival; PFS, progression‐free‐survival; SCLC, small cell lung cancer; TPM, transcript per million.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression in treatment‐emergent prostatic small cell carcinoma. A positive trend in HEPACAM2 ( Left ), SEZ6 ( Middle ), and INSM1 ( Right ) mRNA expression is shown as cancer transitions from adenocarcinoma to adenocarcinoma with NE features to small cell carcinoma of the prostate. The graph displays the mean values as bars, with individual data points representing sample values and error bars indicating the SEM (cBioPortal; metastatic prostate adenocarcinoma; SU2C/PCF Dream Team ²⁵ ). FPKM indicates fragments per kilobase per million; mRNA, messenger RNA; NE, neuroendocrine; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: High HEPACAM2 expression is not associated with other targets for antibody–drug conjugate therapies. Heatmaps showing the relative expression of some common antibody–drug conjugate targets versus HEPACAM2 in two examined SCLC cohorts. The Dowlati cohort represents late‐stage SCLC; George et al. ²⁴ represents early‐stage SCLC. NE indicates neuroendocrine; SCLC, small cell lung cancer.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Functional effects of HEPACAM2 knockdown in vitro. (A) Western blots of identical protein lysates of control and HEPACAM2 knockdown (H2‐1) H69 cells were detected with a ProSci antibody. (B) HEPACAM2 knockdown in H69 cells attenuated mRNA expression of ASCL1 and Myc. The graph displays the mean values as bars, with individual data points representing three independent experiments and error bars indicating the SEM. p < 0.050. gRNA indicates guide RNA; kDa, kilodalton; mRNA, messenger RNA; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Functional Assay, Knockdown, In Vitro, Western Blot, Control, Expressing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Positive correlation between ASCL1 and HEPACAM2. (A) ASCL1 is positively associated with HEPACAM2 on the basis of Pearson analysis. (B) The ENCODE database indicated that ASCL1 binds the HEPACAM2 promoter in several SCLC cell lines. ChIP‐seq indicates chromatin immunoprecipitation sequencing; ENCODE, Encyclopedia of DNA Elements; SCLC, small cell lung cancer.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: ChIP-sequencing

Journal: Cancer

Article Title: Identification of HEPACAM2 as a novel and specific marker of small cell carcinoma

doi: 10.1002/cncr.35557

Figure Lengend Snippet: Functional effects of HEPACAM2 knockdown in SCLC cells. HEPACAM2 knockdown in H1694 cells resulted in a decrease in cell growth as measured by CCK8 proliferation assay up to 5 days (A), a decrease in migration rate as measured by Transwell assay (B), and an increase in cell–cell interaction as indicated by cell attachment counts (C). Results are expressed as mean values, with individual data points representing three independent experiments and error bars indicating the SEM. gRNA indicates guide RNA; SCLC, small cell lung cancer; SEM, standard error of the mean.

Article Snippet: Cells were blocked for 1 h with normal goat serum and incubated overnight at 4°C with HEPACAM2 antibody (0.02 μg/μL; ProSci, 7111).

Techniques: Functional Assay, Knockdown, Proliferation Assay, Migration, Transwell Assay, Cell Attachment Assay