Single cell analysis
Comprehensive analysis of key cancer proteins and pathway markers remains challenging in clinical samples yet is critical in measuring patient response to drugs and in understanding tumor heterogeneity and host response. Currently, the number of protein markers being studied is often limited (<10) in clinical trials and requires time-consuming analyses of tissue sections by specialists. We originally developed a DNA barcoding technology to simultaneously analyze hundreds of proteins in cancer cells harvested from fine needle aspirates, i.e. sampling methods that avoid the morbidity of more invasive core biopsies and potentially allow for more frequent sampling. Compared to existing methods (e.g. immunohistochemistry, cytometry and mass spectrometry), the new DNA-barcoding technology: i) detects hundreds of markers simultaneously, ii) works well in single cells or small numbers of cells, iii) does not destroy valuable samples, iv) is fast and inexpensive and v) can be combined with other genetic analysis techniques (mRNA and DNA analyses). We currently investigate questions such as: How divergent are protein profiles within a malignant lesion (intra-tumor heterogeneity) and between patients (inter-patient heterogeneity)? How do single cell protein levels compare to mRNA levels in the same cells? Does the multiplexed strategy generate unique insights (e.g. signal dynamics, kinetics) that are difficult to assess by conventional approaches?
- Ullal AV*, Peterson V*, Agasti SS, Tuang S, Juric D, Castro CM, Weissleder R. Cancer Cell Profiling by Barcoding Allows Multiplexed Protein Analysis in Fine-Needle Aspirates. Science Transl Medicine. 2014;6(219):219ra9 - PMID: 24431113 - PMCID: PMC4063286
- Peterson VM*, Castro CM*, Chung J, Miller NC, Ullal AV, Castano MD, Penson RT, Lee H, Birrer MJ, Weissleder R. Ascites analysis by a microfluidic chip allows tumor-cell profiling. Proc Natl Acad Sci. 2013;:ePub - PMID: 24297935
Exosome analysis and function
Exosomes (and other extracelluar vesicles, EVs) are shed into circulation by most cancer cells and afford an opportunity to longitudinally study tumor evolution and (non)response to therapies in realtime. Despite the relative abundance of tumor-derived EVs and their informative cargo, one of the major bottlenecks to their use as biomarkers has been diagnostic sensitivity. We have developed a number of different exosome analyses platforms and are focusing on single exosome analysis. In addition to serving as a biomarker discovery platform, analysis of different EVs will provide insight into how many different types of EVs there are based on biogenesis, whether the tumor-derived EV are unique from the normal cell-derived ones and, how they change the phenotype of other normal and tumor cells in support of tumor growth.
- Shao H, Chung J, Balaj L, Charest A, Bigner D, Carter B, Hochberg F, Breakefield X, Weissleder R*, Lee H* (*equal contributions). Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nature Medicine, 2012; 2012;18(12):1835-1840.
- Im H*, Shao H*, Park YI, Peterson VM, Castro CM, Weissleder R*, Lee H*. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nature Biotechnol. 2014;32(5):490-5 - PMID: 24752081
- Shao, H. et al. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat Commun, 2015;6:6999 (2015).
The microbiome and pathogen diagnostics
Our gut and body is colonized by hundreds of microbial species that have important immune modulating functions in health and disease. The composition of the intestinal micro biome has been associated with autoimmune disorders such as rheumatoid arthritis and diabetes, obesity, transplantation, resistance to systemic cancer therapy and the efficacy of immunotherapies. Many antibiotics destroy intestinal microbial communities and increase the susceptibility to deadly pathogens. Furthermore the incriminate use of antibiotics and escalating resistance increases morbidity, mortality, and healthcare costs. While sequencing of bacterial genomes and 16sRNA has enabled modern microbiome research, it is less practical in routine clinical settings where diagnostic results are required in hours. We thus need advanced diagnostics to enable rapid, sensitive, specific, culture-independent species identification of key hospital-associated pathogens. Such advanced diagnostics would i) allow clinicians to quickly determine the most effective treatment for infected individuals, ii) support antimicrobial stewardship by reducing the empiric use of broad-spectrum antimicrobials, and iii) facilitate enrolling target populations in pivotal efficacy trials of new antibiotics, thereby reducing the size and cost of antibacterial clinical trials. We have developed advanced bacterial profiling technologies capable of single bacterial detection sensitivities, genomic phenotyping and measurements of drug resistance genes. We continue to apply them to important clinical and biological questions. We anticipate this new diagnostic technology will enable rapid, culture-independent detection of high-priority antimicrobial-resistant Gram-negative bacterial pathogens and thus address the epidemic of healthcare-associated infections. Some of our technologies have also been commercialized and are FDA approved (T2Biosystems).
- Chung HJ, Castro CM, Im H, Lee H, Weissleder R. A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria. Nature Nanotechnol. 2013;8(5):369-75 - PMID: 23644570 - PMCID: PMC3711657
- Liong M, Hoang AN, Chung J, Gural N, Ford CB, Min C, Shah RR, Ahmad R, Fernandez-Suarez M, Fortune SM, Toner M, Lee H*, Weissleder R* Magnetic barcode assay for genetic detection of pathogens. Nat Commun. 2013;4:1752 - PMID: 23612293 - PMCID: PMC3635151
Immunotherapeutics and innate immune cell function
Tumor-associated macrophages (TAM) are often abundant in the tumor microenvironment and play important roles during tumor spread and in response to therapy. TAM can show considerable plasticity by assuming phenotypes and functions that are either tumoricidal (e.g. M1-like cells) or tumorigenic (e.g. M2-like cells). TAM can also profoundly influence the efficacy of anticancer drugs, nanotherapeutics and immunotherapeutics. Furthermore, TAM have an increasingly well-documented role as relevant therapeutic targets, for example through targeting of the colony-stimulating factor 1 receptor (CSF1R). However, most of our knowledge on TAM and other tumor-associated myeloid cells comes from histological examinations, ex vivo flow cytometry, transcriptome profiling or in vitro culture experiments. There is a significant knowledge gap on how these cells function in vivo. One current project uses high resolution imaging to map TAM subset activity during tumor progression and defines how TAM-targeting agents alter this activity. Related projects are aimed at understanding efficacy or resistance to anti-PD1, anti-PDL1 and anti-CTLA4 therapies.
- Rauch PJ*, Chudnovskiy A*, Robbins CS*, Weber GF, Etzrodt M, Hilgendorf I, Tiglao E, Figueiredo JL, Iwamoto Y, Theurl I, Gorbatov R, Waring MT, Chicoine AT, Mouded M, Pittet MJ, Nahrendorf M, Weissleder R, Swirski FK. Innate Response Activator B Cells Protect Against Microbial Sepsis. Science. 2012;335(6068):597-601; PMID: 22245738, PMCID: PMC3279743
- Swirski FK, Nahrendorf, M, Etzrodt M, Wildgruber M, Cortez-Retamozo V, Panizzi P, Figueiredo JL, Kohler RH, Chudnovkiy A, Waterman P, Aikawa E, Mempel TR, Libby P, Weissleder R, Pittet MJ. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science, 2009; 325:612-26, PMID: 19644120, PMCID: PMC2803111
- Weissleder R, Nahrendorf M, Pittet MJ. Imaging macrophages with nanoparticles. Nat Mater. 2014;13(2):125-38 - PMID: 24452356
We use the term "systems pharmacology" to define research that aims at understanding how drugs work (on specific pathways, on different cell types and in different tissues/organs/diseases), what the variability in patient response is and why many cancer treatments fail. Using a broad array of technologies including in vivo imaging of pharmacokinetics, intravital pharmacodynamic imaging (IPDI), mass spectrometry analysis, novel nanotechnology sensing approaches and chemical biology we obtain quantitative measurements and then develop mechanistic and probabilistic models. Network analysis and quantitative measurements of drug actions and side effects play a key component. Ultimately, we hope to improve our poor understanding of treatment response and define new targets drugs (or synergistic combinations) to tackle complex diseases.
Some of the Questions we are currently addressing are:
- What are the molecular and cellular pathways from drug engaging target to tumor regression for any drug ?
- What makes a cancer cell more, or less, responsive to drug X than a cell in the most relevant normal tissue (where toxicity occurs) ?
- Why do some patients respond better than others? How can we predict the best drug/combination for a particular patient ?
- How do cancers become drug resistant, and how can we combat this ?
- What is the effect of cancer drugs on immune cells and can drug combinations be used to enhance anti-cancer efficacy ?
- What are the most appropriate read-outs of drug efficacy and toxicity in clinical trials ?
- How does taxol work on cancers if only < 5% of cancer cells are in M-phase in vivo ?
This effort is multidisciplinary and inter-institutional involving investigators from the Department of Systems Biology at Harvard Medical School (Initiative in Systems Pharmacology), Harvard affiliated teaching hospitals and MIT.
- Miller MA, Gadde S, Pfirschke C, Engblom C, Sprachman MM, Kohler RH, Yang KS, Laughney AM, Wojtkiewicz G, Kamal N, Bhonagiri S, Pittet MJ, Farokhzad OC, Weissleder R. Predicting therapeutic nano-medicine efficacy using a companion MR imaging nanoparticle. Science Transl Med. 2015;7:314 - PMID: 26582898
- 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. Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug. Nature Commun. 2015;6:8692 - PMID: 26503691
- Laughney AM, Kim E, Sprachman MM, Miller MA, Kohler RH, Yang KS, Orth JD, Mitchison TJ, Weissleder R. Single-cell pharmacokinetic imaging reveals a therapeutic strategy to overcome drug resistance to the microtubule inhibitor eribulin. Science Transl Med. 2014;6 (261):261ra152 - PMID: 25378644
- Thurber GM, Yang KS, Reiner T, Kohler RH, Sorger P, Mitchison T, Weissleder R. Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo. Nature Commun. 2013;4:1504 - PMID: 23422672 - PMCID: PMC3579506