Metabolic Imaging of Head and Neck Cancer Organoids

<div><p>Head and neck cancer patients suffer from toxicities, morbidities, and mortalities, and these ailments could be minimized through improved therapies. Drug discovery is a long, expensive, and complex process, so optimized assays can improve the success rate of drug candidates. This study applies optical imaging of cell metabolism to three-dimensional <i>in vitro</i> cultures of head and neck cancer grown from primary tumor tissue (organoids). This technique is advantageous because it measures cell metabolism using intrinsic fluorescence from NAD(P)H and FAD on a single cell level for a three-dimensional <i>in vitro</i> model. Head and neck cancer organoids are characterized alone and after treatment with standard therapies, including an antibody therapy, a chemotherapy, and combination therapy. Additionally, organoid cellular heterogeneity is analyzed quantitatively and qualitatively. Gold standard measures of treatment response, including cell proliferation, cell death, and <i>in vivo</i> tumor volume, validate therapeutic efficacy for each treatment group in a parallel study. Results indicate that optical metabolic imaging is sensitive to therapeutic response in organoids after 1 day of treatment (p<0.05) and resolves cell subpopulations with distinct metabolic phenotypes. Ultimately, this platform could provide a sensitive high-throughput assay to streamline the drug discovery process for head and neck cancer.</p></div

The redox ratio and fluorescence lifetimes of NAD(P)H and FAD were quantified in organoids treated for 1 day with cetuximab, cisplatin, or their combination.

<p>(A) The redox ratio increases with cetuximab treatment and decreases with cisplatin and the combination treatment. (B) The NAD(P)H lifetime decreases with cetuximab, cisplatin, and combination treatment. (C) The FAD lifetime increases with cetuximab, cisplatin, and combination treatment. *p<0.05, t-test; n~50–200 cells per group.</p

Cellular heterogeneity was analyzed based on NAD(P)H fluorescence lifetime in head and neck cancer organoids after 1 day of treatment with cetuximab, cisplatin, and their combination.

<p>(A) The sum of Gaussian curve fits provides qualitative visualization of cellular heterogeneity. A heterogeneity index (H) indicates low cellular heterogeneity for the control and combination treatment groups compared with higher heterogeneity for the single agent treatment groups. (B) Individual Gaussian curves were plotted and thresholds between the means of the Gaussian curves were color coded to inform spatial mapping, where the red and blue colors represent distinct subpopulations. The total area under the curves is equal across treatment groups. Spatial mapping provides relative locations of cell subpopulations.</p

Untreated organoids and <i>in vivo</i> tumor tissue display distinct optical metabolic imaging properties.

<p>The weighted mean is calculated by τ_m = α₁τ₁ + α₂τ₂, where τ represents the lifetime value and α represents the contribution from each component. (A, C) Organoids exhibit higher NAD(P)H and FAD fluorescence lifetimes (τ_m) compared with <i>in vivo</i> tumor tissue, which is explained by lower contributions of the short lifetime component (α₁), higher values of the short fluorescence lifetime (τ₁), and higher values of the long fluorescence lifetime (τ₂) (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170415#pone.0170415.s001" target="_blank">S1 Fig</a>). (B, D) Population distribution analysis plots cellular heterogeneity for <i>in vivo</i> tumor tissue compared with organoids. The heterogeneity index (H) is similar between organoids and <i>in vivo</i> tumor based on the NAD(P)H fluorescence lifetime and increases for organoids compared to <i>in vivo</i> tumor based on the FAD fluorescence lifetime. The <i>in vivo</i> data is a subset of data published in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170415#pone.0170415.ref028" target="_blank">28</a>]. *p<0.05, t-test, n~100–300 cells per group.</p

Cell proliferation and cell death were quantified using Ki-67 and Cleaved Caspase 3, respectively, in FaDu xenografts after mice were treated for 2 days or 2 weeks, and tumor growth curves show treatment effects over 2 weeks in mice with FaDu xenografts.

<p>(A) Cell proliferation is consistent across treatment groups in FaDu xenografts 2 days after treatment. (B) Cell death increases after 2 days of treatment with cisplatin. (C) Cell proliferation decreases after 2 weeks of treatment with cetuximab, cisplatin, and their combination. (D) Cell death increases after 2 weeks of treatment with cisplatin and the combination of cetuximab and cisplatin. (E) Treatment with cetuximab or cisplatin causes stable disease, whereas combination treatment causes response.*p<0.05 compared with control, rank sum test; †p<0.05, compared with combination treatment, n = 6 tumors.</p

Autofluorescence images show the redox ratio and fluorescence lifetimes of NAD(P)H and FAD in head and neck cancer organoids treated for 1 day with cetuximab, cisplatin, or their combination.

<p>NAD(P)H and FAD autofluorescence images were acquired from the same fields of view, and the redox ratio (top row), NAD(P)H fluorescence lifetime (middle row), and FAD fluorescence lifetime (bottom row) were calculated. For the redox ratio and fluorescence lifetimes, blue represents a low value and yellow represents a high value (see colorbars). Scale bar = 50um.</p

Untreated organoids contain cells with high levels of NAD(P)H intensity and cells with low levels of NAD(P)H intensity.

<p>(A) A representative fluorescence intensity image shows organoids with two levels of NAD(P)H intensity. (B) Low NAD(P)H cells exhibit a lower optical redox ratio than high NAD(P)H cells. (C) Low and High NAD(P)H cells exhibit similar NAD(P)H lifetimes. (D) Low NAD(P)H cells exhibit a higher FAD lifetime than high NAD(P)H cells. Scale bar = 50um. *p<0.05, t-test, n~50–100 cells per group.</p

Metabolic endpoints measure response in SCC25 and SCC61 after treatment.

<p>(a) SCC25 and SCC61 cells were treated with cetuximab, BGT226, or cisplatin for 24 hours. The optical redox ratio is defined as the fluorescence intensity of NADH divided by that of FAD and is normalized by the redox ratio from control cells per day. Treatment with cetuximab does not affect the normalized redox ratio. Treatment with BGT226 or cisplatin decrease the normalized redox ratio. α₁ represents the contribution of the short fluorescence lifetime (free conformation for NADH and protein-bound conformation for FAD) (α₁+α₂ = 1). (b) NADH α₁ decreases after treatment with BGT226 and cisplatin in SCC25 cells and after treatment with cetuximab, BGT226, and cisplatin in SCC61 cells. (c) FAD α₁ decreases after treatment with cisplatin in SCC25 and SCC61 cells. (d) Cells were treated for 24 hours and proliferating cells were labeled with BrdU. The ratio of proliferating cells was calculated by dividing the number of BrdU-labeled cells by the total number of cells per image. Treatment with cetuximab does not affect proliferation. Treatment with BGT226 or cisplatin treatment decrease proliferation. (e) The ratio of lactate production/glucose consumption reflects rates of glycolysis, which decreases after treatment with BGT226 and cisplatin in SCC25 cells and after treatment with cetuximab, BGT226, and cisplatin in SCC61 cells. *p<0.05 rank sum test, compared with control; mean ± SEM.</p

Representative autofluorescence images after treatment.

<p>Representative images of the redox ratio (1st row), NADH α₁ (2nd row), and FAD α₁ (third row) for (a) SCC25 cells and (b) SCC61 cells treated with control (1st column), cetuximab (2nd column), BGT226 (3rd column), or cisplatin (4th column). α₁ quantifies the short lifetime component (α₁+α₂ = 1). NADH α₁ represents the contribution from free NADH, while FAD α₁ conversely represents the contribution from protein-bound FAD. Scale bar represents 30 um.</p

Cyanide treatment alters redox ratio, NADH α₁, and FAD α₁ in nonmalignant oral cells (OKF6).

<p>(a) Cyanide treatment (4 mM) disrupts the electron transport chain, causing an increase in the optical redox ratio. (b) Cyanide treatment increases the contribution of free NADH (α₁) and (c) decreases the contribution of protein-bound FAD (α₁). *p<0.05, rank sum test; mean ± SEM.</p