Biostatistical analysis of quantitative immunofluorescence microscopy images

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.

Download Full-text

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.

Download Full-text