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. 2021 Jan 5;4(1):96–100. doi: 10.1021/acsptsci.0c00156

Single Cell Mass Spectrometry Quantification of Anticancer Drugs: Proof of Concept in Cancer Patients

Ryan C Bensen †, Shawna J Standke †, Devon H Colby †, Naga Rama Kothapalli †, Anh T Le-McClain †, Michael A Patten ‡, Abhishek Tripathi §, Jonathan E Heinlen ∥, Zhibo Yang †,*, Anthony W G Burgett ⊥,*
PMCID: PMC7887743  PMID: 33615163

Abstract

graphic file with name pt0c00156_0004.jpg

In clinical cancer medicine, the current inability to quantify intracellular chemotherapy drug concentrations in individual human cells limits the personalization and overall effectiveness of drug administration. New bioanalytical methods capable of real-time measurement of drug levels in live single cancer cells would allow for more adaptive and personalized administration of chemotherapy drugs, potentially leading to better clinical outcomes with fewer side effects. In this study, we report the development of a new quantitative single cell mass spectrometry (qSCMS) method capable of providing absolute drug amounts and concentrations in single cancer cells. Using this qSCMS system, quantitative analysis of the intracellular drug gemcitabine present in individual bladder cancer cells is reported, including in bladder cancer cells isolated from patients undergoing standard-of-care gemcitabine chemotherapy. The development of single cell pharmacology bioanalytical methods can potentially lead to more effective and safely administered drug medications in patients, especially in the treatment of cancer.

Keywords: single cell mass spectrometry, intracellular drug concentration, patient cells, precision medicine, single probe

New single cell analysis techniques have recently emerged, including DNA and RNA sequencing, microfluidics, imaging, flow cytometry, and mass spectrometry (MS).1−7 The pursuit of single cell mass spectrometry (SCMS) technology has led to the development of new and innovative methodologies utilizing unique apparatuses, ionization technologies, and microscopy.8−13 Research into the treatment and study of cancer is a major potential application of SCMS methods. Understanding abnormal biology at the single cancer cell level is an increasing focus of cancer biology. Cancer stem cell theory and the clinical relevance of circulating tumor cells (CTCs) are examples of the single cell focus of cancer research.14,15

SCMS also has the potential to improve the administration of drug compounds in clinical cancer medicine. The use of SCMS in drug development and in drug administration will be especially important in the emergence of effective precision medicine. Ideally precision medicines in the clinical treatment of cancer should combine new targeted treatments with the capability of assessing, in a time-relevant manner, the efficacy of drug treatments in individual patients.16 The current lack of meaningful, real-time bioanalytical measurements in cancer patient drug administration limits the personalization and responsiveness of the treatment. Due to interindividual pharmacokinetics/pharmacodynamics (PK/PD), the amount of drug present in patient serum often does not report on the amount of drug present at tumor sites.17,18 The measurement of chemotherapy drug concentrations in tissue isolated from tumor sites might not represent the intracellular drug concentration in the tumor cells for multiple reasons.

Tumor tissue samples are usually a complex, heterogeneous mixture of cancer cells and noncancer cells, and multi-drug-resistant (MDR) cancer cells engage P-glycoprotein pumps to extrude therapeutic drugs into the extracellular tumor space.19 Due to these limitations in bioanalysis in drug administration, most chemotherapy drug dosage is determined by a patient’s gross physical characteristics, such as body surface area, rather than any personalized or cancer-specific criteria.20 Further, the chemotherapy drug regimen is commonly administered using a nonadapting, fixed schedule with efficacy determined from an end point analysis, such as an imaging scan (e.g., PET scan), after weeks or months of drug chemotherapy drug administration.21 Patients, therefore, are required to endure extensive, nonpersonalized chemotherapy dosing with its potential associated adverse side effects before any determination if the treatment will have any efficacy.

The development and application of SCMS bioanalytical methods for the detection, quantification, and biological activity of drug compounds would be a powerful tool to provide real-time feedback of therapeutically relevant treatment efficacy.22 Our established single-probe SCMS technology allows for the analysis of small molecules from individual cells, which are attached onto substrate (e.g., glass coverslip) surfaces, under ambient conditions.22,23 Using an isotopically labeled deuterated irinotecan drug compound analogue as an internal standard, we utilized the single-probe system to successfully perform quantitative single cell mass spectrometry (qSCMS) in measuring the intracellular amounts of the anticancer drug irinotecan in single adherent cancer cells.24 To further extend the SCMS analytical methods to nonadherent suspended cells with minimal interferences from complex sampling matrices, we coupled the single-probe device with an integrated cell manipulation platform (ICMP) (Figure 1).25 The ICMP, consisting of an inverted microscope, two cell manipulation systems, a microinjector, and a glass cell-selection probe, is capable of distinguishing cell types, morphologies, and sizes as well as capturing individual, nonadherent cells for analysis.25

Figure 1.

Figure 1

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Quantitative single cell mass spectrometry setup. Schematic of the setup used for quantification of suspended cells utilizing the integrated single-probe/ICMP system.

Here, we report the development of the single-probe/ICMP system to measure the drug concentrations in single nonadherent cells. In addition, using the single-probe/ICMP system, we for the first time performed qSCMS measurement of the amount of a standard-of-care cancer drug gemcitabine in bladder cancer cells isolated from patients undergoing intravenous chemotherapy.

To develop our new method for measuring chemotherapy drugs in patient-isolated cancer cells, we first used the established single-probe qSCMS method to quantify the amount of intracellular gemcitabine inside individual, adherent bladder cancer cells. T24 bladder cancer cells grown in microwells (diameter = 55 μm, depth = 25 μm) on glass chips were treated with either, 0.1, 1.0, or 10 μM gemcitabine for 1 h, followed by single-probe qSCMS analysis (Figure 2, Table 1, and Figure S3). An internal standard of isotopically labeled (13C,15N-labeled) gemcitabine that we prepared ([13CC8H11F215N2NO4 + H]+, m/z 267.0703) (Scheme S1 and Figures S1 and S2) was added into the sampling solvent (acetonitrile with 0.1% formic acid). These results illustrated the ability to detect and quantify the amount of intracellular gemcitabine in individual, adherent bladder cancer cells.

Figure 2.

Figure 2

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Quantification of intracellular gemcitabine. Scatter plot depicting the amount of drug (attomoles) measured at each treatment concentration for each gemcitabine-treated (1 h) cell. (A) T24 cells using the single-probe method and (B) K562 cells using the single-probe/ICMP method (*: significant difference).

Table 1. Quantification of Intracellular Gemcitabine in T24 and K562 Cells.

treatment concentration (μM) T24 (attomole)a K562 (attomole)b
0.1 13.9 ± 10.6 41.3 ± 23.5
1.0 27.0 ± 22.6 46.7 ± 28.2
10 42.8 ± 37.7 72.0 ± 59.5
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a

Adherent cells grown in microwells measured using the single-probe qSCMS method.

b

Suspension cells measured using the single-probe/ICMP method. n > 25 cells for each treatment condition (1 h).

For comparative studies, the single-probe/ICMP setup (Figure 1) was also used to quantify intracellular gemcitabine in detached T24 cells, which were treated for 1 h with 1 μM gemcitabine and trypsinized to release from the solid support. The intracellular amount of gemcitabine in 20 suspended cells was determined to be 37.2 ± 9.2 attomole, which is not significantly different from the value (27.0 ± 22.6 attomole) of the T24 cells while adherent. These results demonstrate the reproducibility of the single-probe/ICMP in suspension cells versus the single probe in adherent cells. Further, K562 suspension leukemia cells were treated with 0.1, 1.0, or 10 μM gemcitabine for 1 h and subjected to the qSCMS measurement using the single-probe/ICMP setup. The intracellular gemcitabine in individual K562 cells followed a similar trend to T24 cells (Figure 2 and Figure S4). The large standard deviation of measured intracellular gemcitabine was likely due to cellular heterogeneity, including broad distribution of cell sizes as well as differences in drug influx, efflux, and conversion.26 Bayesian analyses (Figure S3) indicate that gamma (Γ) distributions can be used to describe intracellular drug levels in both T24 and K562 cell lines, which exhibit a stair-step increase in uptake (isotonic regression). In addition, results from dip test and exact mass test (Figure S3) yield unimodal Γ distribution for both cell lines; that is, there is no clear evidence of the presence of subgroups of intracellular gemcitabine levels based on our current results.

Corresponding to an increase in cellular dose, there is an increase of intracellular gemcitabine in both the adherent T24 cells, measured with the single-probe system, and the suspension K562 cells, measured with the single-probe/ICMP system (Table 1). The increase of intracellular drug amount was not proportional to the increase of compound dosing (i.e., a 10-fold increase in gemcitabine treatment of cells did not produce a 10-fold increase in intracellular gemcitabine levels). Treatment using 10 μM drug compound resulted in a significant increase in the cellular gemcitabine compared to 0.1 μM treatment (Figure 2). Additionally, the spherical, suspended K562 cells allowed for the determination of cellular volume, which was estimated from the diameter of each individual cell using an inverted microscope (Figure 1). With a calculated cell volume, the concentration of gemcitabine in the K562 cells was determined (Table 2). The intracellular concentrations of gemcitabine are significantly increased in a concentration-dependent pattern (Table 2).

Table 2. Concentration of Intracellular Gemcitabine in Suspension K562 Cells Using SCMS and LCMS.

treatment concentration (μM) SCMS (μM)a LCMS (μM)
0.1 10.9 ± 7.1 17.4 ± 5.0
1.0 17.9 ± 11.9 18.1 ± 5.8
10 37.3 ± 26.1 28.0 ± 12.6
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a

n > 20 cells for each treatment condition.

Comparative studies using the traditional LCMS method to measure intracellular drug concentrations in K562 cells were performed. The LCMS analysis was performed on the lysate made from a population of K562 cells. Using the reported cellular volume of an individual K562 cell of 2.8 pL27 and the total cell count, the LCMS results were calculated as the average intracellular drug concentration in single cells. The single-probe/ICMP qSCMS method averaged the results of >20 individual K562 cells. Importantly, the qSCMS single-probe/ICMP method and the LCMS method produced comparable intracellular gemcitabine concentrations in the K562 cells (Table 2).

Having validated the single-probe/ICMP method for gemcitabine qSCMS in the in vitro cancer cell lines, the method was utilized for the analysis of bladder cancer cells isolated from the urine of patients (n = 4). The bladder cancer cells were visually identified in the patient urine based on their clear irregular size and morphological differences from noncancer cells. The single-probe/ICMP system was first used to analyze bladder cancer cells isolated from two patients not undergoing gemcitabine chemotherapy. A series of common cellular species, such as phosphatidylcholines (Figure S5), were detected in both the cancer cell line cells as well as the patient-isolated bladder cancer cells. This consistent detection of biomolecules in the cell lines and patient cancer cells validated the capability of detecting small molecules from clinical cell samples.

Following validation of the single-probe/ICMP method for qualitative analysis in the untreated patients, intracellular gemcitabine quantification was performed on two patients who underwent gemcitabine chemotherapy at a dose of 1000 mg/m2 gemcitabine treatment. Urine was collected from chemotherapy patients 1 h postinfusion (Figure 3 and Figures S6–S10). All qSCMS analyses of patient samples were performed within 3 h of sample generation to limit the possibility of cellular degradation. Detection of gemcitabine in cancer cells isolated from one patient was confirmed through MS/MS analysis in comparison to a standard solution (Figure S9). Gemcitabine was detected only in the cells isolated from the patients who underwent chemotherapy; gemcitabine was not detected in the cells isolated from the untreated patients (Figure 3 and Figures S6 and S7). A gemcitabine quantification data set was compiled consisting of 4–14 successfully measured individual bladder cancer cells per patient infusion (Figure S10). The number of patient cells analyzed was constrained by the 3 h time limit, which included the time required to transport the patient samples for analysis. Additionally, the individual bladder cancer cells had to be identified visually (Figure S9) and captured by the single-probe/ICMP, which was especially time-intensive in urine samples with a relatively low population of bladder cancer cells compared to noncancer cells. The amount of intracellular drug measured from individual cells from patient 1 was (473 ± 188) × 103 and (1.56 ± 1.00) × 103 attomole following the patient’s second and fourth chemotherapy infusions, respectively. Patient 2 had an amount of drug uptake significantly lower than that of patient 1, having 45.8 ± 39.4 and 79.4 ± 50.9 attomole of intracellular gemcitabine following their first and second infusions, respectively. These data suggest that drug uptake efficiency may vary among different patients. Also, multiple infusions could increase the intracellular gemcitabine levels. The measurement of intracellular drug concentration was not conducted due to the inability to accurately measure the dimensions of irregularly shaped patient cancer cells.

Figure 3.

Figure 3

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Gemcitabine spectra from patient 1 isolated bladder cancer cell. Mass spectrum of gemcitabine and the inactive metabolite, dFdU, with 1 μM 15N3-gemcitabine from an individual cell isolated from the urine of a bladder cancer patient 1 h after a 1000 mg/m2 infusion of gemcitabine.

The urine of gemcitabine-exposed patients was also tested by spiking the urine sample with the isotopically labeled gemcitabine as an internal standard (i.e., 100 μM 15N-gemcitabine). Patient 1 had a urine gemcitabine concentration of 1.49 ± 0.47 mM following the fourth infusion, and patient 2 had a urine concentration of 585 ± 159 μM following the first infusion, indicating a relatively large amount of the anticancer compound being excreted through the urine within 1 h after infusion. Relatively higher concentration of gemcitabine in urine from patient 1 might be related to the higher intracellular drug concentration for this patient. Additionally, the nonactive metabolite of gemcitabine, 2′,2′-difluorodeoxyuridine (dFdU) (m/z 265.0621) (Figure 3), was also detected in the patient urine and cancer cells. Detection of dFdU is a potential marker of gemcitabine metabolism and drug inactivation.28

In summary, we have developed the single-probe/ICMP qSCMS system to determine the concentration of chemotherapeutic drugs in single suspended cells in vitro and then used this system to quantify the amount of intracellular gemcitabine in cells isolated from bladder cancer patients. The single-probe/ICMP qSCMS method can be further developed to quantify the amount of many different intracellular molecules of interest (e.g., drug compounds, lipids, metabolites, etc.) and to discriminate cell types based on metabolomic features of individual clinical cells, such as normal versus cancerous cells.29,30 This method can potentially be used to measure therapeutically informative levels of drug compounds within cells in order to develop precision drug monitoring and administration. Single cell quantification of drug compounds isolated from noninvasive patient samples is a major advancement for the development of single cell pharmacology. Single cell pharmacology could lead to understanding the action of drugs on the level of individual disease cells and provide a new paradigm in the development and better administration of drug compounds in patients.

Acknowledgments

This research was supported by grants from the National Institutes of Health (R01GM116116 and R21CA204706) and Barnes Family Foundation (Tulsa, Oklahoma).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.0c00156.

  • Detailed experimental protocols and supplementary figures (PDF)

Author Contributions

▲ (R.C.B.) Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA

Author Contributions

# Ryan Bensen and Shawna Standke are the co-first authors.

The authors declare no competing financial interest.

Supplementary Material

pt0c00156_si_001.pdf (2.6MB, pdf)

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Associated Data

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Supplementary Materials

pt0c00156_si_001.pdf (2.6MB, pdf)

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