Two-photon fluorescence lifetime imaging of intrinsic NADH in three-dimensional tumor models

Anh Cong, Rafaela M.L. Pimenta, Hong Bok Lee, Venkatram R Mereddy, Jon M Holy, Ahmed A Heikal

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like three-dimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior.

Original languageEnglish (US)
Pages (from-to)80-92
Number of pages13
JournalCytometry Part A
Issue number1
StatePublished - Jan 2019

Bibliographical note

Funding Information:
Received 6 June 2018; Revised 6 September 2018; Accepted 11 September 2018 *Correspondence to: Dr. Ahmed A. Heikal, UMD Chemistry and Biochemistry, 1039 University Drive, Duluth, MN 55812. Email: and Dr. Jon Holy, DMED Biomedical Sciences, University of Minnesota Duluth, 1035 University Drive, Duluth, MN 55812. Email: Grant sponsor: University of Minnesota Grant-in-Aid, the Chancellor’s Small Grant, the Department of Chemistry and Biochemistry, the University of Minnesota Duluth (to A. A. H.); Grant sponsor: The Whiteside Foundation, St. Luke’s Hospital, Duluth, MN (to J. H.); Grant sponsor: Science without Borders Program/CNPq (Brazil); Grant number 211897/2014-0; Grant sponsor: Integrated Biosciences Program University of Minnesota Duluth (to R.M.L.P).


  • 3D collagen matrix
  • 4T1
  • FLIM
  • MD1
  • NAD(P)H
  • TPPBr derivative
  • two-photon microscopy

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