Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells

Abstract

Predictions about the cellular efficacy of drugs tested in vitro are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the in vivo cellular response to drugs. This might explain some large discrepancies between in vitro drug studies and drug responses observed in patients. We present a novel in vitro marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed in vitro. The Raman studies detect large panitumumab-induced differences in vitro in cells harboring wild-type K-Ras as seen in A in red but not in cells with K-Ras mutations as seen in B; these studies reflect the observed patient outcomes. However, the effect is not observed when extracellular-signal-regulated kinase phosphorylation is monitored. The Raman spectra show for cells with wild-type K-Ras alterations based on the responses to panitumumab. The subcellular component with the largest spectral response to panitumumab was lipid droplets, but this effect was not observed when cells harbored K-Ras mutations. This study develops a noninvasive, label-free, in vitro vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines. This approach opens a new field of patient-centered drug testing that could deliver superior patient therapies.

Label‐Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches

“…[67] TheR aman study detected large panitumumab-induced differences in cells expressing wild-type K-Ras,b ut not in cells with oncogenic K-Ras mutations.O ther works monitored the effects of the anticancer drug docetaxel on the morphology and biochemistry of living colon cancer cells, [68] the anticancer drug paclitaxel on MCF-7 breast cancer cells, [69] the chemotherapeutic drug doxorubicin on lymphocytes, [70] and the antitumor drugs gemcitabine on living nonsmall lung cancer cells Calu-1 [71] and cisplatin on nasopharyngeal carcinoma cells [72] by Raman microscopic imaging. [67] TheR aman study detected large panitumumab-induced differences in cells expressing wild-type K-Ras,b ut not in cells with oncogenic K-Ras mutations.O ther works monitored the effects of the anticancer drug docetaxel on the morphology and biochemistry of living colon cancer cells, [68] the anticancer drug paclitaxel on MCF-7 breast cancer cells, [69] the chemotherapeutic drug doxorubicin on lymphocytes, [70] and the antitumor drugs gemcitabine on living nonsmall lung cancer cells Calu-1 [71] and cisplatin on nasopharyngeal carcinoma cells [72] by Raman microscopic imaging.…”

Functional link between plasma membrane spatiotemporal dynamics, cancer biology, and dietary membrane-altering agents

“…In addition, increased expression of lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN), lipid-modifying enzymes such as phosphoinositide 3-kinase (PI3K), phospholipase C (PLC) and phospholipase D (PLD) and ATP citrate lyase (ACLY), which collectively promote cholesterol biosynthesis, represent a phenotypic alteration often observed in different cancer types and frequently predict a poor prognosis in cancer patients [199–204]. Furthermore, state-of-the-art analytical and imaging tools, such as electrospray ionization, matrix-assisted laser desorption/ionization, tandem mass spectrometry (MS/MS), and Raman scattering microscopy, have provided valuable lipidomic data that describe alterations in cellular lipid phenotype and fatty acid composition as well as specific spatial cellular distribution in cancer-related conditions [23, 196, 205–208]. It is apparent that cancer cells display a high demand for various lipids and their excess is considered a trademark of cancer.…”

In vitro label‐free screening of chemotherapeutic drugs using Raman microspectroscopy: Towards a new paradigm of spectralomics

“…Vibrational spectroscopy can perform cellular imaging in a (benchtop) microscopic geometry, and can provide non‐invasive label‐free screening of, for example, nanoparticle or drug uptake, trafficking and interaction mechanisms, as well as cellular responses and toxicity , eliminating the need for multiple assays in toxicological screening and drug discovery and preclinical screening stages, by elaborate robotic high‐content analysis systems. Vibrational spectroscopy has also been demonstrated for diagnostic applications, in vivo and ex vivo, in tissue , cells and body fluids .…”

“…For realistic applications potential, however, it is important that these combinatorial signatures are characteristic of the cellular interaction and/or response pathway, and are translatable across cell lines and ultimately patient samples. Although Derenne et al demonstrated that drugs of similar mode of action have similar spectroscopic signatures using FTIR microscopy , there have been few other studies which have attempted to demonstrate consistency between spectroscopic signatures of drug interactions and cellular responses . The identification of specific spectral signatures which are common for drug mechanisms of action and cellular responses opens the perspective to a new “spectralomics” paradigm, in label‐free fingerprinting and monitoring of biological processes in cells in vitro using Raman spectroscopy, with potential applications in fundamental cytological research, pre‐clinical pharmacological development and ultimately improved individualised clinical therapeutics.…”