Use of Quantitative Immunofluorescence Microscopy to Study Intracellular Trafficking

11579 Background: The mechanisms that allow triple negative breast cancer (TNBC) tumors to survive neoadjuvant chemotherapy (NACT) are incompletely understood. Evidence suggests that proliferative heterogeneity may contribute to primary chemotherapy resistance in patients with localized triple negative breast cancer. However, the detailed characterization of a drug-resistant cancer cell state in residual TNBC tissue after NACT has remained elusive. AKT1lowquiescent cancer cells (QCCs) are a quiescent, epigenetically plastic, and chemotherapy resistant subpopulation initially identified in experimental cancer models. Here, we asked whether AKT1low QCCs actually exist in primary tumors from patients with TNBC and persist after treatment with NACT. Methods: We identified QCCs in primary and metastatic human breast tumors using automated, quantitative, immunofluorescence microscopy coupled with computational and statistical analysis. We obtained pre-treatment biopsy, post-treatment mastectomy, and metastatic specimens from a retrospective cohort of TNBC patients treated with neoadjuvant chemotherapy at Massachusetts General Hospital (n = 25). Using automated quantitative immunofluorescence microscopy, QCCs were identified as AKTlow / H3K9me2low / HES1high cancer cells using prespecified immunofluorescence intensity thresholds. QCCs were represented as 2D and 3D digital tumor maps and QCC percentage (QCC-P) and QCC cluster index (QCC-CI) were determined for each sample. Results: We found that QCCs exist as non-random and heterogeneously distributed clusters within primary tumors. In addition, these QCC clusters are enriched after treatment with multi-agent, multi-cycle, neoadjuvant chemotherapy in both residual primary tumors as well as nodal and distant metastases in patients with triple negative breast cancer. Conclusions: Together, these data qualify QCCs as a non-genetic mechanism of chemotherapy resistance in triple negative breast cancer patients that warrants further study.

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Cytokines and oxygen radicals after hyperoxia in preterm and term alveolar macrophages

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00230.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. L1222-L1228 ◽

Cited By ~ 22

Author(s):

Henry J. Rozycki ◽

Paul G. Comber ◽

Thomas F. Huff

Keyword(s):

Inflammatory Cytokine ◽

Immunofluorescence Microscopy ◽

Oxygen Radical ◽

Cytokine Response ◽

Nonspecific Esterase ◽

Rt Pcr ◽

Cytokine Mrna ◽

Quantitative Immunofluorescence ◽

Inflammatory Cytokine Response ◽

Radical Content

To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1β and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2′,7′-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.

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Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training

Physiological Reports ◽

10.14814/phy2.12085 ◽

2014 ◽

Vol 2 (7) ◽

pp. e12085 ◽

Cited By ~ 13

Author(s):

Helen Bradley ◽

Christopher S. Shaw ◽

Philip L. Worthington ◽

Sam O. Shepherd ◽

Matthew Cocks ◽

...

Keyword(s):

Skeletal Muscle ◽

Human Skeletal Muscle ◽

Immunofluorescence Microscopy ◽

Interval Training ◽

Sprint Interval Training ◽

Quantitative Immunofluorescence

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Segregation of Na/H exchanger-3 and Cl/HCO3exchanger SLC26A3 (DRA) in rodent cecum and colon

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00151.2010 ◽

2010 ◽

Vol 299 (2) ◽

pp. G358-G367 ◽

Cited By ~ 58

Author(s):

Colleen Talbot ◽

Christian Lytle

Keyword(s):

Short Chain Fatty Acid ◽

Immunofluorescence Microscopy ◽

Distal Colon ◽

Proximal Colon ◽

Mucosal Surface ◽

Close Proximity ◽

Quantitative Immunofluorescence ◽

Efficient Coupling ◽

Fatty Acid Absorption

The colon is believed to absorb NaCl via the coupled operation of apical Na/H exchanger-3 (NHE3) and Cl/HCO3exchanger SLC26A3 (DRA). Efficient coupling requires that NHE3 and DRA operate in close proximity within common luminal and cytosolic microenvironments. Thus we examined whether these proteins coexist along the apical margin of surface enterocytes by quantitative immunofluorescence microscopy in consecutive colon segments from nonfasted mice and rats. The cecocolonic profiles of NHE3 and DRA expression were roughly inverse; NHE3 was highest in proximal colon (PC) and negligible in distal colon, whereas DRA was absent in early PC and highest in the late midcolon, and DRA was prominent in the cecum whereas NHE3 was not. NHE3 and DRA coexisted only in the middle third of the colon. The consequences of unpaired NHE3/DRA expression on mucosal surface (subscript MS) pH and Na+concentration ([Na+]) were assessed in nonfasted rats in situ using miniature electrodes. In the cecum, where only DRA is expressed, pHMSwas ∼7.5, markedly higher than underlaying stool (6.3), consistent with net HCO3−secretion. In the early PC, where NHE3 is not expressed with DRA, pHMSwas acidic (6.2), consistent with unopposed H+secretion. [Na+]MSwas ∼60 mM in the cecum, decreased along the PC to ∼20 mM, and declined further to ∼10 mM distally. Cl−was secreted into the PC, then reabsorbed distally. Our results suggest a model in which 1) unpaired DRA activity in the cecum maintains an alkaline mucosal surface that could neutralize fermentative H+; 2) unpaired NHE3 activity in the early PC preserves an acidic mucosal surface that could energize short-chain fatty acid absorption; and 3) coupled NHE3/DRA activities in the midcolon allow for vigorous NaCl absorption at a neutral pHMS.

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