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Updated: Apr 12, 2026
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Optimisation of immunofluorescence methods to determine MCT1 and MCT4 expression in circulating tumour cells
Stephen Kershaw1, Jeffrey Cummings2, Karen Morris3
1Clinical and Experimental Pharmacology Group, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. stephen.kershaw-2@manchester.ac.uk.
Background:
The monocarboxylate transporter-1 (MCT1) represents a novel target in rational anticancer drug design while AZD3965 was developed as an inhibitor of this transporter and is undergoing Phase I clinical trials ( http://www.clinicaltrials.gov/show/NCT01791595 ). We describe the optimisation of an immunofluorescence (IF) method for determination of MCT1 and MCT4 in circulating tumour cells (CTC) as potential prognostic and predictive biomarkers of AZD3965 in cancer patients.
Methods:
Antibody selectivity was investigated by western blotting (WB) in K562 and MDAMB231 cell lines acting as positive controls for MCT1 and MCT4 respectively and by flow cytometry also employing the control cell lines. Ability to detect MCT1 and MCT4 in CTC as a 4(th) channel marker utilising the Veridex™ CellSearch system was conducted in both human volunteer blood spiked with control cells and in samples collected from small cell lung cancer (SCLC) patients.
Results:
Experimental conditions were established which yielded a 10-fold dynamic range (DR) for detection of MCT1 over MCT4 (antibody concentration 6.25 microg/mL; integration time 0.4 seconds) and a 5-fold DR of MCT4 over MCT 1 (8 microg/100 microL and 0.8 seconds). The IF method was sufficiently sensitive to detect both MCT1 and MCT4 in CTCs harvested from cancer patients.
Conclusions:
The first IF method has been developed and optimised for detection of MCT 1 and MCT4 in cancer patient CTC.
Insights
An optimized immunofluorescence method detects monocarboxylate transporters (MCT1 and MCT4) in circulating tumor cells (CTCs). This aids in predicting cancer patient response to the MCT1 inhibitor AZD3965.
Area of Science:
- Oncology
- Biomarker Discovery
- Drug Development
Background:
- Monocarboxylate transporter-1 (MCT1) is a target for anticancer drug design.
- AZD3965, an MCT1 inhibitor, is in Phase I clinical trials.
- MCT1 and MCT4 in circulating tumor cells (CTCs) may serve as biomarkers for AZD3965 therapy.
Purpose of the Study:
- To optimize an immunofluorescence (IF) method for detecting MCT1 and MCT4 in CTCs.
- To establish MCT1 and MCT4 as potential prognostic and predictive biomarkers for AZD3965 treatment in cancer patients.
Main Methods:
- Investigated antibody selectivity using western blotting and flow cytometry with cell lines.
- Validated the IF method for detecting MCT1 and MCT4 in CTCs using the Veridex™ CellSearch system.
- Tested the method in spiked blood samples and blood from small cell lung cancer (SCLC) patients.
Main Results:
- Established optimal conditions for MCT1 detection with a 10-fold dynamic range (DR).
- Established optimal conditions for MCT4 detection with a 5-fold DR.
- Demonstrated sufficient sensitivity to detect both MCT1 and MCT4 in patient-derived CTCs.
Conclusions:
- Developed and optimized the first immunofluorescence method for detecting MCT1 and MCT4 in cancer patient CTCs.
- This method provides a tool for biomarker assessment in cancer patients undergoing targeted therapy.
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