Skip to main content

Advertisement

SpringerLink
Log in

TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response

  • Research Article
  • Published:
Molecular Imaging and Biology Aims and scope Submit manuscript

Abstract

Purpose

Personalized medicine has largely failed to produce curative therapies in advanced cancer patients. Evaluation of in situ drug target availability (DTA) concomitant with local protein expression is critical to an accurate assessment of therapeutic efficacy, but tools capable of both are currently lacking.

Procedure

We developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. Using TRIPODD, we demonstrate the feasibility of combining two complementary fluorescence imaging techniques, intracellular paired agent imaging (iPAI) and cyclic immunofluorescence (cyCIF), conducted with oligonucleotide-conjugated antibodies (Ab-oligos) on tissue samples.

Results

We successfully performed sequential imaging on a single tissue section of iPAI to capture single-cell DTA and local protein expression heterogeneity using Ab-oligo cyCIF. Fluorescence imaging data acquisition was followed by spatial registration resulting in high dimensional data correlating DTA to protein expression at the single-cell level where uptake of a targeted probe alone was not well correlated to protein expression.

Conclusion

Herein, we demonstrated the utility of TRIPODD as a powerful imaging platform capable of interpreting tumor heterogeneity for a mechanistic understanding of therapeutic response and resistance through quantification of drug target availability and proteomic response with preserved spatial context at single-cell resolution.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Collisson EA, Cho RJ, Gray JW (2012) What are we learning from the cancer genome? Nat Rev Clin Oncol 9:621–630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Konieczkowski DJ, Johannessen CM, Garraway LA (2018) A convergence-based framework for cancer drug resistance. Cancer Cell 33:801–815

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Garraway LA, Lander ES (2013) Lessons from the cancer genome. Cell 153:17–37

    Article  CAS  PubMed  Google Scholar 

  4. Yap TA, Omlin A, de Bono JS (2013) Development of therapeutic combinations targeting major cancer signaling pathways. J Clin Oncol 31:1592–1605

    Article  CAS  PubMed  Google Scholar 

  5. Lu Y, Ling S, Hegde AM, Byers LA, Coombes K, Mills GB, Akbani R (2016) Using reverse-phase protein arrays as pharmacodynamic assays for functional proteomics, biomarker discovery, and drug development in cancer. Semin Oncol 43:476–483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lin JR, Fallahi-Sichani M, Chen JY, Sorger PK (2016) Cyclic immunofluorescence (CycIF), a highly multiplexed method for single-cell imaging. Curr Protoc Chem Biol 8:251–264

    Article  PubMed  PubMed Central  Google Scholar 

  7. Keren L et al (2019) MIBI-TOF: a multiplexed imaging platform relates cellular phenotypes and tissue structure. Sci Adv 5:eaax5851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Goltsev Y et al (2018) Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell 174:968–981 e915

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Feng Z, Jensen SM, Messenheimer DJ, Farhad M, Neuberger M, Bifulco CB, Fox BA (2016) Multispectral imaging of T and B cells in murine spleen and tumor. J Immunol 196:3943–3950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wagner J et al (2019) A single-cell atlas of the tumor and immune ecosystem of human breast cancer. Cell 177:1330–1345 e1318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Brockman RW (1963) Mechanisms of resistance to anticancer agents. Adv Cancer Res 7:129–234

    Article  CAS  PubMed  Google Scholar 

  12. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674

    Article  CAS  PubMed  Google Scholar 

  13. McAllister SS, Weinberg RA (2010) Tumor-host interactions: a far-reaching relationship. J Clin Oncol 28:4022–4028

    Article  PubMed  Google Scholar 

  14. Marusyk A, Almendro V, Polyak K (2012) Intra-tumour heterogeneity: a looking glass for cancer? Nat Rev Cancer 12:323–334

    Article  CAS  PubMed  Google Scholar 

  15. Keren L et al (2018) A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging. Cell 174:1373–1387 e1319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bunnage ME, Chekler EL, Jones LH (2013) Target validation using chemical probes. Nat Chem Biol 9:195–199

    Article  CAS  PubMed  Google Scholar 

  17. Dubach JM, Kim E, Yang K, Cuccarese M, Giedt RJ, Meimetis LG, Vinegoni C, Weissleder R (2017) Quantitating drug-target engagement in single cells in vitro and in vivo. Nat Chem Biol 13:168–173

    Article  CAS  PubMed  Google Scholar 

  18. Rutkowska A, Thomson DW, Vappiani J, Werner T, Mueller KM, Dittus L, Krause J, Muelbaier M, Bergamini G, Bantscheff M (2016) A modular probe strategy for drug localization, target identification and target occupancy measurement on single cell level. ACS Chem Biol 11:2541–2550

    Article  CAS  PubMed  Google Scholar 

  19. Arrowsmith J, Miller P (2013) Trial watch: phase II and phase III attrition rates 2011-2012. Nat Rev Drug Discov 12:569

    Article  CAS  PubMed  Google Scholar 

  20. Allison M (2012) Reinventing clinical trials. Nat Biotechnol 30:41–49

    Article  CAS  PubMed  Google Scholar 

  21. Miller MA, Zheng YR, Gadde S, Pfirschke C, Zope H, Engblom C, Kohler RH, Iwamoto Y, Yang KS, Askevold B, Kolishetti N, Pittet M, Lippard SJ, Farokhzad OC, Weissleder R (2015) Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug. Nat Commun 6:8692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle II DR, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, Hamann LG (2010) Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465:96–100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Honigberg LA, Smith AM, Sirisawad M, Verner E, Loury D, Chang B, Li S, Pan Z, Thamm DH, Miller RA, Buggy JJ (2010) The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A 107:13075–13080

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cohen MS, Hadjivassiliou H, Taunton J (2007) A clickable inhibitor reveals context-dependent autoactivation of p90 RSK. Nat Chem Biol 3:156–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Stadler C, Rexhepaj E, Singan VR, Murphy RF, Pepperkok R, Uhlén M, Simpson JC, Lundberg E (2013) Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nat Methods 10:315–323

    Article  CAS  PubMed  Google Scholar 

  26. Simon GM, Niphakis MJ, Cravatt BF (2013) Determining target engagement in living systems. Nat Chem Biol 9:200–205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fischman AJ, Alpert NM, Rubin RH (2002) Pharmacokinetic imaging: a noninvasive method for determining drug distribution and action. Clin Pharmacokinet 41:581–602

    Article  CAS  PubMed  Google Scholar 

  28. Lomenick B, Hao R, Jonai N, Chin RM, Aghajan M, Warburton S, Wang J, Wu RP, Gomez F, Loo JA, Wohlschlegel JA, Vondriska TM, Pelletier J, Herschman HR, Clardy J, Clarke CF, Huang J (2009) Target identification using drug affinity responsive target stability (DARTS). Proc Natl Acad Sci U S A 106:21984–21989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Matthews PM, Rabiner EA, Passchier J, Gunn RN (2012) Positron emission tomography molecular imaging for drug development. Br J Clin Pharmacol 73:175–186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Martinez Molina D et al (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341:84–87

    Article  PubMed  CAS  Google Scholar 

  31. Munteanu B, Meyer B, von Reitzenstein C, Burgermeister E, Bog S, Pahl A, Ebert MP, Hopf C (2014) Label-free in situ monitoring of histone deacetylase drug target engagement by matrix-assisted laser desorption ionization-mass spectrometry biotyping and imaging. Anal Chem 86:4642–4647

    Article  CAS  PubMed  Google Scholar 

  32. Budayeva HG, Kirkpatrick DS (2020) Monitoring protein communities and their responses to therapeutics. Nat Rev Drug Discov 19:414–426

    Article  CAS  PubMed  Google Scholar 

  33. Grimwood S, Hartig PR (2009) Target site occupancy: emerging generalizations from clinical and preclinical studies. Pharmacol Ther 122:281–301

    Article  CAS  PubMed  Google Scholar 

  34. Schurmann M, Janning P, Ziegler S, Waldmann H (2016) Small-molecule target engagement in cells. Cell Chem Biol 23:435–441

    Article  PubMed  CAS  Google Scholar 

  35. Pressman D, Day ED, Blau M (1957) The use of paired labeling in the determination of tumor-localizing antibodies. Cancer Res 17:845–850

    CAS  PubMed  Google Scholar 

  36. Baeten J, Haller J, Shih H, Ntziachristos V (2009) In vivo investigation of breast cancer progression by use of an internal control. Neoplasia 11:220–227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Liu JT et al (2009) Quantifying cell-surface biomarker expression in thick tissues with ratiometric three-dimensional microscopy. Biophys J 96:2405–2414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pogue BW, Samkoe KS, Hextrum S, O’Hara JA, Jermyn M, Srinivasan S, Hasan T (2010) Imaging targeted-agent binding in vivo with two probes. J Biomed Opt 15:030513

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Davis SC, Gibbs SL, Gunn JR, Pogue BW (2013) Topical dual-stain difference imaging for rapid intra-operative tumor identification in fresh specimens. Opt Lett 38:5184–5187

    Article  PubMed  PubMed Central  Google Scholar 

  40. Tichauer KM, Deharvengt SJ, Samkoe KS, Gunn JR, Bosenberg MW, Turk MJ, Hasan T, Stan RV, Pogue BW (2014) Tumor endothelial marker imaging in melanomas using dual-tracer fluorescence molecular imaging. Molec Imaging Biol 16:372–382

    Article  Google Scholar 

  41. Tichauer KM, Diop M, Elliott JT, Samkoe KS, Hasan T, Lawrence KS, Pogue BW (2014) Accounting for pharmacokinetic differences in dual-tracer receptor density imaging. Phys Med Biol 59:2341–2351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tichauer KM, Samkoe KS, Gunn JR, Kanick SC, Hoopes PJ, Barth RJ, Kaufman PA, Hasan T, Pogue BW (2014) Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging. Nat Med 20:1348–1353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Barth CW, Schaefer JM, Rossi VM, Davis SC, Gibbs SL (2017) Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery. Theranostics 7:4722–4734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Maniwa Y, Yoshimura M, Obayashi C, Inaba M, Kiyooka K, Kanki M, Okita Y (2001) Association of p53 gene mutation and telomerase activity in resectable non-small cell lung cancer. Chest 120:589–594

    Article  CAS  PubMed  Google Scholar 

  45. Tichauer KM, Samkoe KS, Sexton KJ, Gunn JR, Hasan T, Pogue BW (2012) Improved tumor contrast achieved by single time point dual-reporter fluorescence imaging. J Biomed Opt 17:066001

    Article  PubMed  PubMed Central  Google Scholar 

  46. Tichauer KM, Samkoe KS, Sexton KJ, Hextrum SK, Yang HH, Klubben WS, Gunn JR, Hasan T, Pogue BW (2012) In vivo quantification of tumor receptor binding potential with dual-reporter molecular imaging. Molec Imaging Biol 14:584–592

    Article  Google Scholar 

  47. Tichauer KM, Holt RW, el-Ghussein F, Davis SC, Samkoe KS, Gunn JR, Leblond F, Pogue BW (2013) Dual-tracer background subtraction approach for fluorescent molecular tomography. J Biomed Opt 18:16003

    Article  PubMed  CAS  Google Scholar 

  48. Wang D, Chen Y, Leigh SY, Haeberle H, Contag CH, Liu JTC (2012) Microscopic delineation of medulloblastoma margins in a transgenic mouse model using a topically applied VEGFR-1 probe. Transl Oncol 5:408–414

    Article  PubMed  PubMed Central  Google Scholar 

  49. Meng B, Folaron MR, Byrd BK, Samkoe KS, Strawbridge RS, Barth C, Gibbs SL, Davis SC (2020) Topical dual-probe staining using quantum dot-labeled antibodies for identifying tumor biomarkers in fresh specimens. PLoS ONE 15:e0230267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. House BJ, Schaefer JM, Barth CW, Davis SC, Gibbs SL (2019) Diagnostic performance of receptor-specific surgical specimen staining correlate with receptor expression level. Proceedings of SPIE--the International Society for Optical Engineering 10862

  51. Folaron MR, Strawbridge RR, Samkoe KS, Gibbs SL, Davis SC (2019) Effect of staining temperature on topical dual stain imaging of tissue specimens for tumor identification. Proceedings of SPIE--the International Society for Optical Engineering 10862, 108620 L

  52. Schaefer JM, Barth CW, Davis SC, Gibbs SL (2019) Diagnostic performance of receptor-specific surgical specimen staining correlates with receptor expression level. J Biomed Opt 24:1–9

    Article  PubMed  Google Scholar 

  53. McMahon NP, Jones JA, Kwon S, Chin K, Nederlof MA, Gray JW, Gibbs SL (2020) Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence for future use in clinical histopathology. J Biomed Opt 25:1–18

    Article  PubMed  Google Scholar 

  54. Saka SK, Wang Y, Kishi JY, Zhu A, Zeng Y, Xie W, Kirli K, Yapp C, Cicconet M, Beliveau BJ, Lapan SW, Yin S, Lin M, Boyden ES, Kaeser PS, Pihan G, Church GM, Yin P (2019) Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues. Nat Biotechnol 37:1080–1090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ullal AV et al (2014) Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates. Sci Transl Med 6:219ra219

    Article  CAS  Google Scholar 

  56. Decalf J, Albert ML, Ziai J (2019) New tools for pathology: a user’s review of a highly multiplexed method for in situ analysis of protein and RNA expression in tissue. J Pathol 247:650–661

    Article  PubMed  Google Scholar 

  57. Wang Y, Woehrstein JB, Donoghue N, Dai M, Avendaño MS, Schackmann RCJ, Zoeller JJ, Wang SSH, Tillberg PW, Park D, Lapan SW, Boyden ES, Brugge JS, Kaeser PS, Church GM, Agasti SS, Jungmann R, Yin P (2017) Rapid sequential in situ multiplexing with DNA exchange imaging in neuronal cells and tissues. Nano Lett 17:6131–6139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Johnson LN (2009) Protein kinase inhibitors: contributions from structure to clinical compounds. Q Rev Biophys 42:1–40

    Article  CAS  PubMed  Google Scholar 

  59. McMahon NP et al (2020) Fluorescent imaging for in situ measurement of drug target engagement and cell signaling pathways. Proc SPIE Int Soc Opt Eng 11219:112190O

    PubMed  PubMed Central  Google Scholar 

  60. Solanki A, Wang L, Korber J, McMahon N, Tichauer K, Samkoe KS, Gibbs SL (2020) Intracellular paired agent imaging enables improved evaluation of tyrosine kinase inhibitor target engagement. Proc SPIE Int Soc Opt Eng 11219:112190F

  61. Chang YH et al. (2017) In 2017 39th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) 672-675

  62. Berg S, Kutra D, Kroeger T, Straehle CN, Kausler BX, Haubold C, Schiegg M, Ales J, Beier T, Rudy M, Eren K, Cervantes JI, Xu B, Beuttenmueller F, Wolny A, Zhang C, Koethe U, Hamprecht FA, Kreshuk A (2019) ilastik: interactive machine learning for (bio)image analysis. Nat Methods 16:1226–1232

    Article  CAS  PubMed  Google Scholar 

  63. Roma-Rodrigues C, Mendes R, Baptista PV, Fernandes AR (2019) Targeting tumor microenvironment for cancer therapy. Int J Mol Sci 20:840

    Article  CAS  PubMed Central  Google Scholar 

  64. Li H, Takayama K, Wang S, Shiraishi Y, Gotanda K, Harada T, Furuyama K, Iwama E, Ieiri I, Okamoto I, Nakanishi Y (2014) Addition of bevacizumab enhances antitumor activity of erlotinib against non-small cell lung cancer xenografts depending on VEGF expression. Cancer Chemother Pharmacol 74:1297–1305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Yu HA, Schoenfeld AJ, Makhnin A, Kim R, Rizvi H, Tsui D, Falcon C, Houck-Loomis B, Meng F, Yang JL, Tobi Y, Heller G, Ahn L, Hayes SA, Young RJ, Arcila ME, Berger M, Chaft JE, Ladanyi M, Riely GJ, Kris MG (2020) Effect of osimertinib and bevacizumab on progression-free survival for patients with metastatic EGFR-mutant lung cancers: a phase 1/2 single-group open-label trial. JAMA Oncol 6:1048–1054

    Article  PubMed  Google Scholar 

  66. Cannon TM, Shah AT, Skala MC (2017) Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures. Biomed Opt Express 8:1911–1925

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Walsh AJ, Cook RS, Sanders ME, Aurisicchio L, Ciliberto G, Arteaga CL, Skala MC (2014) Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer. Cancer Res 74:5184–5194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Dudenkova VV, Shirmanova MV, Lukina MM, Feldshtein FI, Virkin A, Zagainova EV (2019) Examination of collagen structure and state by the second harmonic generation microscopy. Biochemistry (Mosc) 84:S89–S107

    Article  CAS  Google Scholar 

  69. Burke RM, Madden KS, Perry SW, Zettel ML, Brown EB 3rd (2013) Tumor-associated macrophages and stromal TNF-alpha regulate collagen structure in a breast tumor model as visualized by second harmonic generation. J Biomed Opt 18:86003

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Drs. Stefanie Kaech Petrie and Crystal Chaw as well as Mr. Brian Jenkins in the Advanced Light Microscopy at the Jungers Center at Oregon Health and Science University (OHSU) for expert technical assistance with fluorescence microscopy studies.

Funding

This work was funded by an ASPIRE Award from the Mark Foundation for Cancer Research (Gibbs).

Author information

Authors and Affiliations

  1. Biomedical Engineering Department, Oregon Health & Science University, Portland, OR, USA

    Nathan P. McMahon, Allison Solanki, Lei G. Wang, Antonio R. Montaño, Jocelyn A. Jones & Summer L. Gibbs

  2. Thayer School of Engineering at Dartmouth College, Dartmouth College, Hanover, NH, USA

    Kimberley S. Samkoe

  3. Department of Surgery, Geisel School of Medicine at Dartmouth College, Dartmouth College, Hanover, NH, 03755, USA

    Kimberley S. Samkoe

  4. Department of Biomedical Engineering, Illinois Institute of Technology, Chicago, IL, 60616, USA

    Kenneth M. Tichauer

  5. Knight Cancer Institute, Oregon Health & Science University, Collaborative Life Sciences Building, 2730 S Moody Ave, Mail Code: CL3SG, Portland, OR, 97201, USA

    Summer L. Gibbs

Authors
  1. Nathan P. McMahon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Allison Solanki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lei G. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonio R. Montaño
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jocelyn A. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kimberley S. Samkoe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kenneth M. Tichauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Summer L. Gibbs
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Summer L. Gibbs.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

McMahon, N.P., Solanki, A., Wang, L.G. et al. TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response. Mol Imaging Biol 23, 650–664 (2021). https://doi.org/10.1007/s11307-021-01589-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11307-021-01589-x

Key words

Search

Navigation