Circulating Tumor Cells

One of the proposed mechanisms of cancer metastasis is the dissemination of tumor cells from the primary organ into the blood stream. A cellular link between the primary malignant tumor and the peripheral metastases has been established in the form of CTCs in peripheral blood. While extremely rare (1 in 10 billion cells), these cells provide a potentially accessible source for early detection, characterization and monitoring of cancers that would otherwise require invasive serial biopsies. The emerging fields of medical technology and microfluidics offer a radically different approach to rare cell detection, which is particularly relevant to the isolation of CTCs. Working in collaboration with Drs. Shyamala Maheswaran and Daniel Haber, we have designed a microfluidic device, the CTC-Chip, that allows the isolation and characterization of CTCs from the peripheral blood of cancer patients. The chip design was centered on the concept of passive mixing of blood through the generation of microvortices, ultimately improving the capture of rare cells by dramatically increasing the number of interactions between the target CTCs and the antibody-coated substrate. Using blood from patients with metastatic and localized cancer, we have demonstrated the ability to isolate, enumerate and molecularly characterize putative CTCs with high sensitivity and specificity. Additionally, microclusters of CTCs have been captured in a rare number of patient samples. These clusters of CTCs present an intriguing phenomenon; however their significance has yet to be determined. Ongoing projects include translating the technology for early cancer detection, exploring these clusters of CTCs, increasing capture sensitivity through amplification of cell surface antigens, and the design of biomaterials for the release of the rare cells from the device surface.

 
Photos by Felice Frankel

 

Cell Capture with Nano-Porous Structures

State-of-the-art in vitro diagnostics and separation techniques exploit highly specific biomolecular recognition processes for surface capturing of targets (biomarker proteins, pathogens, tumor cells, etc.) from a heterogeneous mixture. Although these in vitro techniques present unprecedented opportunities, their potential is often hindered by the inefficient delivery of the targets to the ligands immobilized on solid surfaces. This fundamental limitation, known as the mass transport problem, plagues even the most advanced detection tools with single molecule sensitivities under medically relevant conditions of low target concentrations. To overcome mass transport limitations, we have recently proposed fluidically active nanoporous surfaces enabling diversion of streamlines towards the capturing surface while promoting reaction kinetics due to low shear stress. Using our platform, we demonstrated two orders of magnitude improved capturing efficiencies of circulating tumor cells from blood samples compared to earlier schemes.



Non-conventional Approaches for CTC Capture

We are also developing alternative approaches for capturing circulating tumor cells (CTCs) in blood utilizing specific cell-cell interactions. As-formed cell clusters may play a significant role in promoting cancer metastasis. The images show a cluster of PC-3 cancer cells, platelets, and leukocytes that were captured in a microfluidic device.



Whole Blood Preservation for In-Vitro Diagnostics

We are working on whole blood preservation ex vivo for a variety of clinical diagnostic applications. The image shows microfluidic capture of circulating tumor cells from blood that was preserved for two days at room temperature. The project draws together the expertise in biopreservation and microfluidic diagnostics within the CEM, as well as the MGH Cancer Center.



High-content and high-throughput imaging of cancer cells

Cancer cells can be highly heterogeneous, with rare metastasis precursors capable of giving rise to a metastatic lesion mixed in with other tumor cells undergoing apoptosis. Thus, due to this heterogeneity, quantitative, robust analysis for individual cells may be critical for determining a particular cancer cells’ their clinical relevance in different disease contexts. Due to limitations in the number of distinct spectra that can be used in wide-field fluorescence imaging, high throughput characterization of cells and tissue is traditionally done with three to four colors. Our lab is exploring alternative imaging modalities, such as multi-spectral imaging (MSI), to enable quantitative analysis of multiple (8+) markers on a single cell. Our interest in MSI is driven by the technology’s capability to image as many colors as distinct antibodies available and by dramatic reductions in sample autofluorescence. We are also interested in using this technology to develop an automated system to quantitatively analyze RNA-ISH and FISH signals in cells and tissue.

 

Capturing CTCs with low surface marker expression

Some CTCs express low amounts of the surface markers that are used to distinguish cancer cells from other cells in blood. We are working on ways to amplify these surface markers to improve cell capture efficiency. The image shows a low surface marker-expressing cancer cell, along with some other cell types. The different colors in the image allow us to distinguish cancer cells from other cell types.



References

  1. Stott S.L.*, Lee R.J.*, Nagrath S.*, Yu M., Miyamoto D.T., Ulkus L., Inserra E.J., Ulman M., Springer S., Nakamura Z., Moore A.L.,Tsukrov D.I., Kempner M.E., Dahl D.M., Wu C.L., Iafrate A.J., Smith M.R., Tompkins R.G., Sequist L.V., Toner M., Haber D.A., Maheswaran S. Isolation and characterization of circulating tumor cells from patients with localized metastatic prostate cancer. Science Translational Medicine 2010; 2(25):25. *co-authors
  2. Stott S.L.*, Hsu C.-H.*, Tsukrov D.I., Yu M., Miyamoto D.T., Waltman B.A., Rothenberg S.M., Shah A.M., Smas M.E., Korir G.K., Floyd Jr. F.P., Gilman A., Lord J.B., Winokur D., Springer S., Irimia D., Nagrath S.N., Sequist L.V., Lee R.J., Isselbacher K.J., Maheswaran S., Haber D.A., Toner M., “Isolation of circulating tumor cells using a microvortex-generating herringbone-chip,” Proc Natl Acad Sci. 107 (43), 18392-19397, 2010.* co-authors.
  3. Yu M., Stott S.L., Toner M., Maheswaran S., Haber D.A., “Circulating Tumor Cells: Approaches to Isolation and Characterization” Journal Cell Biology 192 (3), 373-382, 2011.
  4. Shah A.M., Yu M., Nakamura Z., Ciciliano J., Ulman M., Kotz K.T., Stott S.L., Maheswaran S., Haber D.A., Toner M., “A Biopolymer System for Cell Recovery from Microfluidic Cell Capture Devices” Analytical Chemistry 84 (8), 3682–3688, 2012.
  5. Yu M.*, Ting D.T.*, Stott S.L., Wittner B.S., Ozsolak F., Paul S., Ciciliano J.C., Smas M.E., Gilman A.J., Ulman M.J., Contino G., Alagesan B., Brannigan B.W., Milos P.M., Ryan D.P., Sequist L.V., Bardeesy N., Ramaswamy S., Toner M., Maheswaran S.*, Haber D.A.* “RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis” Nature, 487 (7408), 510-513, 2012 *co-authors
  6. Miyamoto D.T.*, Lee R.J.*, Stott S.L.*, Ting D.T., Wittner B.S., Ulman M., Smas M.E., Lord J.B., Brannigan B.W., Tratuwein J., Bander N.H., Wu C.L., Sequist L.V., Smite M.R., Ramaswamy S., Toner M., Maheswaran S., Haber D.A., “Androgen receptor signaling in circulating tumor cells as a marker of hormonally responsive prostate cancer” Cancer Discovery, Oct23 epub, 2012 *co-authors
  7. Yu M.*, Bardia A.*,Wittner B.S., Stott S.L., Smas M.E., Ting D.T., Isakoff S.J., Ciciliano J.C., Wells M.N., Shah A.M., Concannon K.F., Donaldson M.C., Sequist L.V., Brachtel E., Sgroi D., Baselga J., Ramaswamy S., Toner M., Haber D.A., Maheswaran S., “Circulating Breast Tumor Cells in Humans Exhibit Dynamic Changes in Epithelial and Mesenchymal Cell Composition” Science, 339 (6119), 2013 *co-authors