EP3210024A1 - Materials and methods for assaying living cells - Google Patents
Materials and methods for assaying living cellsInfo
- Publication number
- EP3210024A1 EP3210024A1 EP15852876.0A EP15852876A EP3210024A1 EP 3210024 A1 EP3210024 A1 EP 3210024A1 EP 15852876 A EP15852876 A EP 15852876A EP 3210024 A1 EP3210024 A1 EP 3210024A1
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- EP
- European Patent Office
- Prior art keywords
- cells
- cancer
- tumor
- substrate
- tumor cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
Definitions
- the field of the invention relates to the fields of cancer, particularly methods of diagnosis/prognosis and treatment of metastatic cancer and materials and devices useful for the same.
- Circulating tumor cells are shed from a primary tumor into the bloodstream. Such cells can nucleate new tumor masses at sites distant to the primary tumor.
- CTCs Circulating tumor cells
- the presence of CTCs in a cancer patient's blood is associated with a poorer disease prognosis, and methods are available to measure the number CTCs in a cancer patient's bloodstream.
- CTCs are usually present in very small numbers, and some patients in whom CTCs are not detected eventually do develop tumor metastases. Accordingly, new methods are needed for identifying patients at risk for developing tumor metastases.
- Microtentacles are microtubule-based protrusions of the membrane of certain types of tumor cells.
- Microtubules composed of detyrosinated alpha-tubulin (Glu-tubulin) or acetylated tubulin (Ace-tubulin) have vastly increased stability in vivo, persisting for hours rather than the three to five minutes observed for microtubules composed of tyrosinated tubulin (Tyr- tubulin).
- Microtentacles are supported by stable microtubules, enriched with Glu-tubulin or Ace-tubulin. Up- regulation of the intermediate filament vimentin is another mechanism by which tumor cells stabilize microtubules.
- Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.
- Cell 158: 1110-1122. Accordingly, the presence of microtentacles on the surface of a tumor cell indicates a poorer patient prognosis.
- Circulating tumor cells can lead to metastatic recurrence but the effects of current cancer drugs on the dynamic responses of these free-floating tumor cells is almost completely overlooked.
- CTCs Circulating tumor cells
- Persistent cell cycle arrest renders CTCs highly resistant to traditional chemotherapies targeting cell growth or division (Naumov, G.N., et al., Cancer Res, 2002. 62: 2162-8; Naumov, G.N., et al., Breast Cancer Res Treat, 2003. 82: 199-206).
- Free-floating tumor cells produce microtentacles that promote metastatic reattachment.
- mammary epithelial and breast carcinoma cells produce extensions of the plasma membrane when detached from extracellular matrix (ECM) (Whipple, R.A., et al., Cancer Res, 2008. 68: 5678-88; Whipple, R.A., A.M. Cheung, and S.S. Martin, Exp Cell Res, 2007. 313: 1326-36) (See Fig. 1). These extensions occur with higher frequency in metastatic breast tumor cell lines (Whipple, R.A., et al., Cancer Res, 2008. 68: 5678-88).
- Microtentacles can encircle adjacent cells promoting reattachment of tumor cells to each other and extracellular matrix (Whipple, R.A., et al., Cancer Res, 2008. 68: 5678-88; Whipple, R.A. et al., Exp Cell Res, 2007. 313: 1326-36), but are not observed when cells are grown attached to matrix. There is currently an overwhelming focus of the tumor biology field on cells that are attached to flattened or 3-dimensional extracellular matrix proteins (Yamaguchi, H., J. Wyckoff, and J. Condeelis, Curr Opin Cell Biol, 2005. 17: 559-64; Fischbach, C, et al., Nat Methods, 2007.
- Taxol strongly promote McTNs (Balzer, E.M., et al., Breast Cancer Res Treat, 2010.
- Neoadjuvant drug effects on CTCs could be increasing metastatic risk. While the concentration of Taxol (1.2 ⁇ ) that promotes McTNs is higher than that used to inhibit cell division, it is worth noting that a routine clinical dose (175mg/m 2 ) yields blood levels of >60 ⁇ Taxol that do not decrease below 1.2 ⁇ until 6 hours following infusion (Bulitta, J.B., et al., Cancer Chemother Pharmacol, 2009. 63: 1049-63). Since CTCs travel from primary tumors to distant tissues within minutes (Chambers, A.F., A.C. Groom, and I.C. MacDonald, Nat Rev Cancer, 2002.
- Neoadjuvant Taxol treatment also increases CTCs more than 1000- fold (Pachmann, K., et al., J Clin Oncol, 2008. 26: 1208-15) when used in advance of surgery. Since neoadjuvant taxane treatment is growing more common (Gralow, J., et al., Clin Breast Cancer, 2008. 8: 33-7), these results emphasize the importance of determining how an individual patient's tumor cells respond to neoadjuvant chemotherapy. During neoadjuvant therapy, a subset of patients unfortunately can show rapid progression (Caudle, A.S., et al., J Clin Oncol, 2010.
- McTNs promote aggregation and endothelial attachment of free-floating tumor cells.
- the inventor's lab determined that the delicate structure of McTNs required new methods to image dynamic shape changes in live, detached tumor cells (Balzer, E.M., et al., Oncogene, 2010. 29: 6402- 8; Balzer, E.M., et al., Breast Cancer Res Treat, 2010. 121 : 65-78.; Whipple, R.A., et al., Cancer Research, 2008. 68: 5678-88).
- McTNs encircle neighboring cells to promote the aggregation of detached tumor cells (Matrone, M.A., et al., Cancer Res, 2010. 70: 7737-41) (See Fig. 6).
- Such resolution of individual McTNs would be impossible with traditional fixed-cell immunofluorescence or even electron microscopy, since surface or cytoskeletal stains cannot distinguish between the neighboring cells (See Fig. 6A).
- McTNS promote tumor cell clustering is particularly notable since CTCs clusters have recently been shown to have 50-fold higher metastatic potential (Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA et al (2014). Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158: 1110-1122.).
- microtentacles When freshly isolated from a solid tumor, generate long and dynamic microtubule-driven protrusions of the plasma membrane after dissociation and detachment referred to herein as microtentacles.
- the present invention is directed to methods and compositions for detecting microtentacles in cellular samples to improve prognosis and provide for more effective cancer therapies by screening drugs for their ability to inhibit or promote microtentacle formation or stability.
- the present invention provides a device for assaying living cells, comprising a substrate, wherein the substrate comprises one or more tethering molecules, which adhere to the substrate and are capable of interacting with cell membranes of the cells, wherein the cells maintain a free-floating, non-adherent character when bound to the one or more tethering molecules.
- the invention provides a device which tethers the membrane of living cells, such as tumor cells, to an imaging surface that is exceptionally thin (lOnm-lOOnm) and optically clear. Tethered tumor cells continue to move dynamically as if they are free-floating, because the cell membrane is only held at a small point.
- the invention enables a new microfluidic slide to capture and image microtentacles on patient tumor cells to determine their responses to cytoskeletal cancer drugs.
- the invention provides a perspective on how patient tumors respond to drug treatments (e.g., in less than 24 hours).
- the invention tests FDA-approved drugs. The findings can be rapidly translated to clinical treatment.
- the invention can establish which immediate microtentacles response of the tumor cells to drugs predict metastasis in mouse tumorgrafts (which is a known indicator of eventual patient metastasis).
- the device provides a platform that can be used for testing patient tumors and to improve the understanding of how to select cancer therapies that reduce metastatic risk.
- the invention provides a microfluidic device to enable the rapid imaging of free-floating tumor cells removed from cancer patients (e.g., breast, prostate, colon, lymphoma, or other cancer types) during surgery or core needle biopsy, which provides an opportunity to advance clinical imaging of live patient tumor cells.
- cancer patients e.g., breast, prostate, colon, lymphoma, or other cancer types
- the invention provides a new microfluidic device to tether fresh patient tumor cells to a patterned surface for microtentacle imaging with confocal microscopy.
- the cell-tethering array slide will only require -200 tumor cells or less from patients, an amount easily obtainable from surgical samples or even core needle biopsies. Moreover, this cell-tethering array will hold cells but prevent cell adherence to the surface so that the dynamic behavior of microtentacles continue.
- the invention provides a method of making a device for assaying living cells, comprising:
- a substrate with one or more layers of one or more materials that substantially inhibit the cells from adhering to the substrate; and ii) contacting the coated substrate with one or more tethering molecules, which adhere to the substrate and are capable of interacting with cell membranes of the cells, wherein the cells maintain a free-floating, non-adherent character when bound to the one or more tethering molecules.
- the invention provides methods to identify a patient with an increased likelihood of having or developing metastatic cancer.
- tumor cells from a tumor or tumor biopsy removed from the patient are analyzed to determine whether the tumor cells, when dissociated from the tumor and detached from substrate, display microtentacles on their cell surface. The presence of microtentacles on the surface of the tumor cells identifies a patient with an increased likelihood of having or developing metastatic cancer.
- the present invention also provides methods to rapidly determine how a patient's tumor will respond to various cancer drugs, by incubating the above- mentioned tumor cells with various cancer drugs while detached from substrate, to determine whether the drugs decrease the incidence of microtentacles on the surface of the tumor cells.
- drugs that increase the number and/or length of microtentacles on the surface of the tumor cells should not be administered to the patient, whereas cells that decrease (or at least do not increase) the number and/or length of microtentacles on the surface of the tumor cells can be administered to the patient.
- a therapy which increases microtentacles could be used in combination with a therapy that reduces microtentacles to counteract the detrimental effects of microtentacle promotion.
- the invention provides a method for imaging microtentacles on isolated, living primary tumor cells from a cancer subject, comprising:
- the invention provides a method of identifying a subject with an increased likelihood of having or developing metastatic cancer comprising:
- the invention provides a method for determining whether a candidate drug inhibits or promotes microtentacle formation and/or stability on isolated, living primary tumor cells from a cancer subject comprising: i) obtaining one or more living primary tumor cells that has been isolated from a solid tumor from the subject;
- the invention provides a method for determining the stem cell potential of tumor cells from a cancer subject comprising:
- the invention provides a method for imaging microtentacles on isolated, living, non-adherent primary tumor cells from a cancer subject comprising:
- the invention provides a method of identifying a subject with an increased likelihood of having or developing metastatic cancer comprising: i) obtaining one or more living non- adherent primary tumor cells that has been isolated from a solid tumor from the subject;
- the invention provides a method for determining whether a candidate drug inhibits or promotes microtentacle formation and/or stability on isolated, living, non-adherent primary tumor cells from a cancer subject comprising: i) obtaining one or more living, non-adherent primary tumor cells that has been isolated from a solid tumor from the subject; ii) contacting the one or more living, non-adherent primary tumor cells with the candidate drug;
- the invention provides a method for determining the stem cell potential of tumor cells from a cancer subject comprising:
- FIG. 1 Microtentacles in metastatic breast tumor cells.
- McTNs the delicate microtubule structure of McTNs causes them to collapse when treated with traditional fixatives (methanol, formaldehyde), which is a major reason why these structures were previously overlooked.
- traditional fixatives methanol, formaldehyde
- B cell surface glycosylation
- C lipophilic membrane dyes
- FIG. 2 The microtubule- stabilizing Tau protein is elevated in metastatic tumors and increases lung retention of injected tumor cells.
- FIG. 3 Cancer drugs can mimic the cytoskeletal effects of wound healing and detachment.
- Epithelial cells at the wound edge also undergo an epithelial-to-mesenchymal transition (EMT) that induces increased stem cell characteristics.
- EMT epithelial-to-mesenchymal transition
- FIG. 4 Targeting cell division can increase McTNs.
- FIG. 5 Poor recurrence-free survival when CTCs increase during neoadjuvant chemotherapy. Patients whose CTC levels decrease during neoadjuvant chemotherapy have a 100% recurrence-free survival after 7 years (upper line). In stark contrast, patients whose CTC levels increase during neoadjuvant therapy have only a 4% recurrence-free survival after 7 years (lower line).
- McTNs promote tumor cell aggregation.
- McTNs promote penetration of endothelial monolayers.
- FIG. 8 Breast tumor stem cells have elevated microtentacles that promote reattachment.
- A) Flow cytometry sorting of immortalized human mammary epithelial (HMEC) cells for those that express high levels of CD44 and low levels of CD24 (CD44+/CD24-) isolates a stem cell population from breast tumor cell lines.
- FIG. 9 Live patient tumor cells show evidence of McTNs.
- Epithelial organoids derived from patient breast tumors were dissociated and viewed live with confocal microscopy after membrane labeling with CellMask.
- Low (A) and high (B,C) magnification confocal images show evidence of McTNs (white arrows).
- D microtentacle extension is also evident (D).
- FIG. 10 Surgical samples from breast cancer patients (membrane dynamics - 600x)
- FIG. 11 Drug responses can be measured quickly in patient-derived tumor cells (30 minute Colchicine response)
- FIG. 12 Automated Measurements of Microtubule (McTN) Characteristics.
- FIG. 13 McTNs in prostate tumor cell lines. LnCAP, DU145 and PC3 prostate tumor cells were imaged for McTNs with CellMask confocal microscopy (upper panels) or DIC brightfield microscopy (lower panels). Weakly metastatic LnCAP cells have a smooth surface and very few McTNs, while the more highly metastatic DU145 and PC3 cells have more McTNs. Automated McTN analysis and published stem cell markers in prostate cancer are shown for each cell line.
- FIG. 14 Schematic showing tethering of cells to a microfluidic slide to image free floating patient cancer cells.
- the tether can hold the cell in place for confocal imaging and efficient capture of images.
- FIG. 15 Confocal images of tethered cells treated with paclitaxel and cholchicine.
- FIG. 16 Schematic showing isolation of cancer cells and subsequent analysis of microtentacles using a microfluidic device and tumor graft growth and metastasis.
- FIG. 17 Polyelectrolyte multilayer (PEM) films as ultrathin surface coatings.
- C) Coatings can be assembled from synthetic or natural components, allowing tuning of CTC adhesion by modifying PEMs with DNA or lipids.
- FIG. 18 DNA-lipid tethering of breast tumor cells maintains free-floating cell behavior.
- A) The DNA labeling technique can be used to pattern CTCs on planar surfaces), creating a platform to monitor McTN dynamics in arrayed cells (B) and the impact of drug exposure (C) in real- time.
- FIG. 19 PEM allows tuning of tumor cell adhesion and support maintenance of McTNs.
- FIG. 20 Testing adhesion of patient-derived cell mixtures in microfluidic slides. Microtentacles are observable in patient tumor cells (arrows, left), but samples include non-tumor cell types (right).
- FIG. 21 Commercially-available glycerophospholipids (Avanti Polar Lipids, Inc.) for integration into a polyelectrolyte layer.
- FIG. 22 ⁇ allows patterning of single-cell lipid tethers on a cytophobic PEM background.
- FIG. 23 Photoactivatable lipid anchor for lithography directly in microfluidic channels.
- FIG. 24 Automated McTN analysis. MDA-436 cells were treated with vehicle (0.1 DMSO) or colchicine (50 ⁇ ) or paclitaxel ( ⁇ ) and imaged for McTNs with confocal microscopy. A gradient vector flow snake algorithm was used to define the cell edges and a local curvature maxima effectively identified McTN ends (yellow diamonds). Quantitation of at least 10 random cells shows that Colchicine (Col) significantly reduces the average number of McTNs per cell, while paclitaxel (Tax) increases McTNs, but this does not reach significance in MDA-436 cells. This automated cell shape analysis will also enable calculations of average McTN length, curvature, etc. that will yield more multi-dimensional data for McTN studies.
- Col Colchicine
- Tax paclitaxel
- FIG. 25 PEMs form a cytophobic layer on microfluidic device, allowing for McTN visualization while maintaining optical clarity.
- A Schematic depicting coating of Ibidi micro-slide with PEMs to promote free floating cells for McTN visualization.
- B PEM coatings increase in size (left axis, black) with increasing number of bilayers while maintaining optical clarity (right axis, gray) Data correspond to the mean of samples prepared in triplicate with error bars representing SEM.
- C Representative maximum intensity z-projection of MDA-MB-436 cells on PEM coated slides showing McTNs (arrows). Scale bar represents ⁇ .
- FIG. 26 Lipid-tethered PEMs, while optically clear, allow for tethering of cells to slides for visualization.
- A Schematic depicting how lipids deposited as the terminal layer of a PEM coated slide promotes interactions with cell membranes.
- B Film thickness and
- C optical clarity measurements with the addition of lipids DOPC or DOTAP (Stott SL, et al. (2010) Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences 107(43): 18392-18397). Lipids promote growth of film while maintaining an optically clear substrate for imaging. Data correspond to the mean of samples prepared in triplicate where three measurements were taken per slide with error bars representing SEM.
- FIG. 27 DOTAP can tether breast cancer cell lines after washing. Percent cell retention of (A) MDA-MB-436 and (B) MCF-7 cells plated on micro-slides coated with 4 PMA/PAAm bilayers alone or with DOTAP and allowed to tether for 1 hour. The remaining cells after each wash were quantified with CellProfiler and expressed as a percent of the initial cell number. Percent cell retention of (C) MDA- MB-436 and (D) MCF-7 cells plated on micro-slides coated with 4 PMA/PAAM bilayers alone or with DOPC and allowed to tether for 1 hour. The remaining cells after each wash was quantified with CellProfiler and expressed as a percent of the initial cell number.
- FIG. 28 Lipid tethering retains free-floating characteristics of breast tumor cells and does not affect cell viability.
- A McTN quantification of MDA-MB-436 and MCF-7 cells suspended on a low-attach plate, micro-slides with PEM-no tether, and micro-slides with PEM-DOTAP tether. Data represents blinded quantification of McTN frequency from three independent experiments with 100 cells counted for each (mean +/- SEM).
- B Representative images of McTNs (arrows) on MDA-MB-436 cells seeded PEM-no tether and PEM-DOTAP tether micro-slides at 40x magnification. Scale bar represents 10 ⁇ .
- Viability of (C) MDA-MB-436 and (D) MCF-7 cells calculated at 0 and 6 hours after seeding on micro-slides with PEM- DOTAP tether. Fluorescence of live and dead cells were quantified for each (according to green or red fluorescence) and divided by total cell number to quantify percent of live and dead cells, respectively using CellProfiler. Data represents mean cell viability from three independent experiments (mean +/- SEM).
- E Representative images show viability of MDA-MB-436 cells tethered by DOTAP for 6 hours. Phase contrast images show total cell number, live, and dead at 4x magnification. Scale bar represents 200 ⁇ .
- FIG. 29 Lipid tethering allows for real-time microtentacle imaging in response to drug treatment and minimizes effects of drift.
- A Microtentacle (arrows) imaging of MDA-MB-436 cells seeded on micro-slides with PEM-no tether (i-iii) and PEM-DOTAP tether (iv-vi). Representative ⁇ slice (i and iv), maximum z- projection of 5 slices at one time point (ii and v), and maximum t-projection after 20 frames (iii and vi) are shown at 60x magnification.
- FIG. 30 PEM prevents attachment of MDA-MB-436 and MCF-7 breast cancer cells.
- FIG. 31 PEM prevents cell attachment and does not affect cell viability.
- FIG. 32 PEM does not affect viability of MDA-MB-436 and MCF-7 cells.
- Phase contrast images show total cell number, live (green fluoresce), and dead (red fluorescence).
- Phase contrast images show total cell number, live and dead (green and red fluorescence) at 4x magnification. Scare bar represents 200 ⁇ .
- FIG. 33 Triton-X is a positive control for cell death.
- FIG. 34 DOTAP can tether MCF-7 breast tumor cells. Representative images of MCF-7 cells seeded on micro-slides with PEM-no tether, PEM-DOTAP tether, and PEM-DOPC tether prior to washing and after 3 subsequent washes at 4x magnification. Scale bar represents 200 ⁇ .
- FIG. 35 Lipid tethering retains microtentacles and does not affect cell viability.
- A Representative image of McTNs (arrows) on MDA-MB-436 cells on a low-attach plate at 40x magnification. Scale bar represents ⁇ .
- B Representative images of McTNs (arrows) on MCF-7 cells on a low-attach plate, micro-slide with PEM-no tether, and micro-slide with PEM-DOTAP tether at 40x magnification. Scale bar represents ⁇ .
- C Representative images of MCF-7 cells seeded on micro- slides with PEM-DOTAP tether for 6 hours. Phase contrast images show total cell number, live and dead cells at 4x magnification. Scale bar represents 200 ⁇ .
- FIG. 36 Quantitative image analysis of time-dependent McTN metrics.
- B) Analyzing cells at 10s timepoints allows the dynamic movement of McTNs under vehicle-treated conditions to be appreciated (blue lines).
- FIG. 37 Tracking individual McTN tip motion. Applying a particle-tracking algorithm to the McTN tips identified in Fig. 36 allows the movement of individual McTN tips (color- coded) to be followed over time. Taxol has clear effects to reduce average McTN speed and make McTNs more persistent (reduces the area over which a McTN moves).
- FIG. 38 PEM coating for McTN studies.
- A) Blinded McTN scoring demonstrates that McTN frequencies remain the same for cells regardless of whether they are suspended over low-attach plates (published method), PEMs, or PEMs with an integrated DOTAP cell tether.
- FIG. 39 Photo-crosslinkable custom lipid tether.
- FIG. 40 Patterned arrays of tethered tumor cells facilitate imaging cell groups at high magnification without clustering (A), mechanical measurements (B) or systematic testing of cell-cell contact distance (C). In addition, the ability to track the fates of individual cells over time will allow us to determine if stem cells are selected or converted by drug treatments (D).
- the present invention generally concerns the previously unknown phenomenon that certain primary tumor cells isolated from a patient's solid tumor and placed under non-adherent in vitro culture conditions can generate microtubule protrusions that increase the cells' ability to reattach to each other and/or surfaces. Because tumor cells become detached during spread through the blood or lymphatics, the generation of these protrusions is important for the ability of the tumor cells to spread metastatically to distant tissues and/or organs. Thus, the ability of a tumor cell to form such microtubule protrusions is an indication of its metastatic potential. In vivo, such microtubule protrusions can act to enhance tumor cell adhesion to vessel walls and/or allow tumor cells to avoid being crushed by size-restriction in capillaries.
- protrusions increase in number and size per cell in more metastatic tumor cell lines. Protrusions also occur with a significantly higher frequency in populations of breast tumor cells with greater metastatic potential. In particular, death of cancer patients is most often caused by metastatic spread of the primary tumor through the bloodstream. However, large tumor cells are efficiently killed by shearing when they are pushed through small-diameter capillaries by blood pressure. The microtubule protrusions can help metastatic tumor cells avoid death by adhering to vessel walls and/or bracing against them before the size of the capillary becomes limiting.
- the invention focuses on the imaging and detection of these structures in living, non-adherent primary tumor cells isolated from a solid tumor from a cancer subject to improve prognosis and provide for more effective cancer treatments.
- the invention relates to materials and devices that are useful for assaying living cells, such as tumor cells.
- microtentacles in tumor cells taken directly from tumors, from tumor cell biopsies or primary tumors surgically removed from cancer patients.
- tumor cells with microtentacles were present only as circulating tumor cells in the bloodstream of cancer patients having tumor metastases or at risk for metastases.
- the present discovery allows direct examination of cells from a primary tumor or surgical biopsy for the presence of microtentacles, which has various benefits that provide prognostic information and can guide patient treatment.
- the present discovery provides an immediate functional phenotype to measure (as opposed to a static gene or protein expression profile).
- the tumor cell imaging studies can be immediately conducted with very few cells. Because no long-term cell culturing is needed, the risk of in vitro selection for tumor cells with altered properties relative to the patient's original tumor cells is avoided.
- the inventor has discovered that tumor stem cells are enriched in microtentacles at their cell surface. Thus, analysis of a patient's fresh tumor cells can provide an indication of a tumor's "sternness," which is known to be associated with an increased metastatic risk.
- microtentacle dynamics are very rapid, drug effects can be measured within minutes.
- the imaging of microtentacles on the surface of tumor cells taken from tumor biopsies or surgically-removed tumor specimens provides the opportunity to rapidly gauge a tumor's potential response to various cancer drugs prior to treatment, thereby allowing treatment to be tailored to the patient.
- the present invention further provides a new device (for example, a microfluidic cell tethering slide) to rapidly image cytoskeletal dynamics in free-floating patient tumor cells to better understand how individual patient tumors respond to cancer drugs.
- the device can also be used for research purposes on cultured cells.
- MRI/PET-CT magnetic resonance imaging methods
- doctors are currently unable to accurately follow early metastasis.
- most cancer drug development and clinical trials are aimed at reducing the growth of these large tumors, very little is known about how current cancer therapies are influencing tumor metastasis.
- microtentacles refers to extensions of the plasma membrane in detached cells that are enriched in tubulin protein, for example Glu- tubulin or Ace-tubulin and largely devoid of polymerized actin.
- tubulin protein for example Glu- tubulin or Ace-tubulin
- enhancement of the protrusions such as with the protrusions increasing in length, number per cell and frequency in a population, for example. This is in contrast to well-known invadopodia and podosomes associated with adherent tumor cells that are actin-based and inhibited by actin depolymerization.
- non-adherent primary tumor cells are cells that have been isolated from a solid tumor by human intervention and placed under in vitro conditions in which the cells generally do not form significant protein-based contacts to an in vitro surface (such as that of a coated or uncoated cell culture chamber or microfluidic slide) during the time in which the tumor cells are being tested or studied, so as to allow the formation of microtentacles by the tumor cells capable of forming such micro tentacles.
- Such "non-adherent" cells include cells that are tethered to the in vitro surface, e.g., by an oligonucleic acid tether or lipid tether as described herein.
- cells "isolated from a solid tumor” means cells are isolated from a solid tumor by human intervention and do not encompass detached cancer cells that have escaped their primary organ site, and are present elsewhere in the body of the patient, for example in the bloodstream or lymph nodes.
- the cells that are isolated from a solid tumor are primary cells, and do not encompass cells from cancer cell lines, which were originally derived from a solid tumor.
- the cells are isolated directly from a non-removed tumor, from a tumor cell biopsy, from circulating tumor cells or from a tumor that is surgically removed.
- the isolated cells are grown in mice (tumorgrafts, PDX) and then analyzed.
- the isolated cells are cultured with conditional reprogramming and then analyzed.
- the invention provides a device for assaying living cells, comprising a substrate, wherein the substrate comprises one or more tethering molecules which adhere to the substrate and are capable of interacting with cell membranes of the cells, wherein the cells maintain a free-floating, non-adherent character when bound to the one or more tethering molecules.
- the invention provides a method of making a device for assaying living cells, comprising: i) coating a substrate with one or more layers of one or more materials that substantially inhibit the cells from adhering to the substrate; and ii) contacting the coated substrate with one or more tethering molecules which adhere to the substrate and are capable of interacting with cell membranes of the cells, wherein the cells maintain a free-floating, non-adherent character when bound to the one or more tethering molecules.
- the substrate is coated with one or more materials to minimize adhesion of cells or tissue to the substrate.
- the materials are polymeric materials.
- the polymeric material is polyethylene oxide (PEO) and copolymers thereof.
- the polymeric materials are selected from PEO-methacrylate-PVC copolymer and polyethylene oxide -polypropylene copolymers.
- the polymeric materials are cationic and/or anionic polymers.
- the substrate is coated with polyelectrolyte multilayer films (PEMs).
- PEMs polyelectrolyte multilayer films
- PEM films provide a tunable surface for tethering and imaging cells. Adsorption of alternating layers of polycationic and polyanionic solutions to surfaces can be used to generate polyelectrolyte multilayers (PEM) that assemble through electrostatic or hydrogen bonding (Fig. 17A).
- these films are ultrathin (10-lOOnm), optically clear, and use a simple all-aqueous approach based on sequential exposure of a target surface or substrate to the polyelectrolyte solutions.
- Fig. 17B This feature allows simple deposition of films on complex surface geometries, including microfluidic channels. Furthermore, this platform allows assembly from a robust combination of synthetic polymers and biological molecules such as nucleic acids, lipids, proteins (Fig. 17C).
- electrolyte as used herein means any chemical compound that ionizes when dissolved.
- polyelectrolyte as used herein means a polymeric electrolyte, such as polyacrylic acid.
- multilayer means a structure comprised of two or more layers.
- polyelectrolyte films may be deposited on a surface, via the repetitive, sequential adsorption from aqueous solution of oppositely charged polyelectrolytes.
- such films may be deposited on a surface via the repetitive, sequential adsorption from dilute aqueous solution of polymers, of which at least one polymer is a polyelectrolyte, comprising complementary hydrogen-bond donor functionality or hydrogen-bond acceptor functionality or both.
- any synthetic or natural polyion can be used to coat the substrate to create polyelectrolyte multilayer films (PEMs) using an aqueous based process.
- the resulting polyelectrolyte multilayers can coat substrates of any size or shape with defined properties of film thickness, composition, conformation, roughness, and wettability.
- the layers can be deposited in a pH-dependent manner. See, e.g., WO 2003/035278 Al.
- PEMs are prepared by adsorption of alternating layers of polycationic and polyanionic aqueous solutions to the substrate that assemble through electrostatic or hydrogen bonding.
- some aspects of the methods for making the device of the invention comprise coating a substrate, comprising sequentially depositing on a substrate, alternating layers of polymers to provide a coated surface, wherein a first polymer is selected from one or more cationic polyelectrolytes and a second polymer is selected from one or more anionic polyelectrolytes, thereby preventing cell adhesion to said coated surface.
- the cationic polymer material is not limiting.
- the polycationic material can be any water-soluble polycationic polymer.
- Representative polycationic materials include natural and unnatural polyamino acids having net positive charge at neutral pH, positively charged polysaccharides, and positively charged synthetic polymers.
- suitable polycationic materials include polyamines having amine groups on either the polymer backbone or the polymer sidechains, such as poly-L-lysine and other positively charged polyamino acids of natural or synthetic amino acids or mixtures of amino acids, including poly(D-lysine), poly (ornithine), poly(arginine), and poly(histidine), and nonpeptide polyamines such as poly(aminostyrene), poly(aminoacrylate), poly (N-methyl aminoacrylate), poly (N- ethylaminoacrylate), poly(N,N- dimethyl aminoacrylate), poly (N,N- diethylaminoacry late), poly(aminomethacrylate), poly (N-methyl amino- methacrylate), poly(N- ethyl aminomethacrylate), poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethyl aminomethacrylate), poly(ethyleneimine), polymers of quaternary amines, such as
- the polycationic material is used to form at least one of the multilayers.
- the cationic polymer is polyallylamine hydrochloride (PAH).
- the cationic polymer is polyacrylamide (PAAm).
- the anionic polymer material is not limiting.
- the polyanionic material can be any biocompatible water-soluble polyanionic polymer. Suitable materials include alginate, carrageenan, furcellaran, pectin, xanthan, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate, poly(meth)acrylic acid, oxidized cellulose, carboxymethyl cellulose and crosmarmelose, synthetic polymers and copolymers containing pendant carboxyl groups, such as those containing maleic acid or fumaric acid in the backbone. Polyaminoacids of predominantly negative charge are also suitable.
- the anionic polymer is polyacrylic acid (PAA). In some embodiments, the anionic polymer is polymethacrylic acid (PMA). In some embodiments, anionic polymer is poly(styrene sulfonate) (SPS).
- This layer-by-layer deposition process provides a means to create polycation- polyanion polyelectrolyte multilayers one molecular layer at a time, thereby allowing control over the composition and surface functionality.
- alternate layers of positively and negatively charged polymers are sequentially adsorbed onto a substrate from dilute solution to build up interpenetrated multilayer structures.
- the PEMs comprise alternating layers of the cationic polymer polyacrylamide (PAAm) and the anionic polymer polymethacrylic acid (PMA). In some embodiments, the PEMs comprise alternating layers of poly-L-lysine (PLL) and poly-L-glutamic acid (PGA).
- the thickness of the coating is not limiting provided that it is effective to minimize adhesion of cells or tissue to the substrate. In some embodiments, the coating has a thickness that is less than 1 nm. In some embodiments, the thickness can range from about 1 nm to about 500 nm.
- the thickness can range from about 1 nm to about 450 nm, from about 3 nm to about 250 nm, from about 5 nm to about 150 nm, from about 10 nm to about 100 nm, and from about 10 nm to about 50 nm.
- the PEMs have a thickness of from about 10 nm to about 100 nm.
- the substrate is coated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more bilayers. In some embodiments, the substrate is coated with 1-10, 2-10, 2-6, or 3-5 bilayers.
- the thickness of the coating and number of layers of applied material is such that the coated substrate is substantially optically clear in order to enable visualization of the cells, e.g., using microscopy, such as confocal microscopy.
- the optical clarity defined by percent transmittance of light, is greater than about 70%. In some embodiments, the optical clarity is greater than about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.
- the substrate of the device comprises one or more tethering molecules which adhere to the substrate and are capable of interacting with cell membranes of cells.
- the device comprises PEMs that incorporate tethering molecules in their top surface.
- the tethering molecules hold the cells in a substantially fixed position, so that the cells can be visualized by microscopy, such as confocal microscopy.
- the tether molecule is a hydrocarbon. In some embodiments, the tether molecule is a lipid. In some embodiments the tether molecule, such as a lipid, has a charged headgroup to support adherence to the substrate. In some embodiments, the interaction between the tether molecule and the substrate is an electrostatic interaction. In some embodiments, for efficient cell tethering, a lipid with long, hydrophobic fatty acids is required to associate with the cell membrane. In some embodiments, the lipid has at least 9 carbon atoms and is capable of associating with the plasma membrane of cells.
- the lipid has at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms. In some embodiments, the lipid has from 9-25, 10-20, 12- 19, or 13-18 carbon atoms. In some embodiments, the charged lipid can be added to the top layer of the coated substrate, such as the PEM film within a microfluidic channel slide.
- Charged lipids that are suitable for use as tethers include, but are not limited to, glycerophospholipid, phospholipids, phosphatidylcholine, phosphatidic acid, lysophosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidylinositolphosphates, bis(monacylglycero)phosphate, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-dioleoyl-i «-glyceio-3-phosphocholine (DOPC), 18:0-LysoPG and 15:0(3)-16:1-CA, salts and dervivatives thereof. Suitable charged lipids are also commercially available (see Avanti Polar Lipids, Inc.).
- the tether molecule is DOTAP.
- a charged lipid such as DOTAP is used as a tether on substrates comprising a PMA/PAAm bilayer.
- a negatively or positively charged headgroup in the lipid can interact with the surface of the PMA/PAAm bilayer (Fig. 21).
- a negatively charged phosphate in each glycerophospholipid can be used to interact with the positively charged amide group in the terminal PAAm layer of the PEM film.
- lipids can be prepared for adsorption using the lipid film rehydration technique.
- lipid ratios (1 :10-10:1 LysoPG:CA) at total concentrations of 1 - 10 ⁇ of lipid can be dried under nitrogen, then sonicated ( 12W, 30 sec) in 500 ⁇ of HEPES. The PEM-coated surface or channel can then be exposed to the lipid solution for 5 minutes and washed twice.
- the tethering molecule such as a charged lipid
- the crosslinking agent that can be used is not limiting and can include chemical crosslinking agents or photo-crosslinking agents.
- Chemical crosslinking agents are well known and can include, for example, formaldehyde, ethylene glycol diacrylate, di(ethylene glycol) diacrylate, tetra(ethylene glycol) diacrylate, ethylene glycol dimethacrylate, di(ethylene glycol) dimethacrylate, tri(ethylene glycol) dimethacrylate, ⁇ , ⁇ '-Methylenebisacrylamide, ⁇ , ⁇ '- Methylenebisacrylamide, ⁇ , ⁇ ' -( 1 ,2-Dihydroxyethylene)bisacrylamide, N-(l - Hydroxy-2,2-dimethoxyethyl)acrylamide, and divinylbenzene.
- Conjugation of a photo-cleavable charged group to the end of the tether molecule can generate a molecule that can be integrated in solution to the coated substrate.
- the tether comprises a photoactivatable group that can be crosslinked to the substrate.
- the tether is crosslinked to reactive amines that are present on the substrate coating, such as PMA/PAAm bilayers.
- the tethering molecules are organized into an array pattern on the substrate. When the cells are bound, it will create an organized array of cells which will facilitate screening methods. In some embodiments, the tethering molecules are organized into an array pattern on the substrate, wherein the pattern array comprises islands of from about 4-15 ⁇ in size with about 25-100 ⁇ center spacing. In some embodiments, the tethering molecules are organized into an array pattern on the substrate, wherein the pattern array comprises islands of about 7 ⁇ in size with about 40 ⁇ center spacing.
- the tethering molecules are organized into an array pattern on the substrate by a process comprising microcontact printing.
- an array of single-cell lipid attachment points can be made using a PDMS microcontact printing ( ⁇ ) procedure that patterns lipid on the upper PEM layer (e.g., PAAm or PLL) in small 5-10 ⁇ spots (See Kohli et ai, J Colloid Interface Sci. 301: 461-469 (2006)).
- the tethering molecules are organized into an array pattern on the substrate by a process comprising photo-crosslinking of the tether to the substrate at defined sites.
- a lipid tether comprises an aryl azide group that can be photo-crosslinked to the substrate (FIG. 39).
- the lipid tether comprises a 6-nitropiperonyloxymethyl (NPOM) group, which can be coupled to lipid via an amine linker and then cleaved through illumination with UV light at 365nm (FIG. 23). The charges present on the NPOM can reduce hydrophobic interactions with the cell membrane.
- cells are added to the device and bound to the tethering molecules. The cells can also be fixed to the substrate prior to or following the analysis. In some embodiments, the cells are fixed with a chemical fixative, such as formaldehyde.
- the properties or characteristics of the cells that can be assayed are not limiting. Once the cells have been added to the device, they can be assayed for one or more properties of interest. In some embodiments, the cells are assayed for the presence, size, stability, frequency, formation, and inhibition of microtentacles. In some embodiments, tumor cells are assayed for the presence of microtentacles, using one or more techniques such as confocal microscopy. In some embodiments, tumor cells are assayed for their responsiveness to drug treatment, including promotion or inhibition of microtentacles. In some embodiments, the stem cell potential of tumor cells from a cancer subject is assayed according to their ability to display microtentacles and/or form stem cell spheres.
- the responses of tethered cells can be used to select optimal drug treatments. For example it may be desirable in some instances to promote more stem cell characteristics in certain cell types (e.g., wound healing, aging diseases, immunodeficiencies). In some embodiments, various cell types and drugs can be screened and selected that promote more stem cell characteristics such as increased microtentacles/spheres.
- the cells that can be assayed are not limiting.
- the cells are non-adherent cells.
- Various types of non-adherent cells are described in the literature and are well known. See, e.g., J Immunol Methods. 1983 Jul 15;61(2): 145- 50.
- the cells to be assayed are selected from the group consisting of blood cells, bone marrow cells, lymph cells, stem cells, oocytes, muscle cells, epithelial cells and tumor cells.
- the cells to be assayed are tumor cells.
- the tumor cells are from a cancer selected from the group consisting of breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, brain cancer, liver cancer, testicular cancer, thyroid cancer, skin cancer, colon cancer, ovarian cancer, cervical cancer, and uterine cancer.
- the tumor cells are primary tumor cells that have been isolated from a solid tumor from a subject.
- the tumor cells are isolated directly from a non-removed tumor, from a tumor cell biopsy, from circulating tumor cells or from a tumor that is surgically removed.
- the tumor cells comprise one or more micro tentacles.
- the device is a microfluidic device.
- Microfluidics generally refers to systems, devices, and methods for processing small volumes of fluids. These types of systems and devices are well known in the art. Because microfluidic systems can process a wide variety of fluids, such as chemical or biological samples, these systems have many application areas, such as biochemical assays (for, e.g., medical diagnoses), biochemical sensors, etc.
- One type of microfluidic device is a microfluidic chip.
- Microfluidic chips can include micro-scale features (or "microfeatures”), such as channels, valves, pumps, and/or reservoirs for storing fluids, for routing fluids to and from various locations on the chip, and/or for reacting fluidic reagents.
- microfluidic chips can include more complex micro-scale structures such as mixing devices or sensors for performing other processing functions on the fluids.
- the microfluidic device of the invention comprises one or more channels.
- the device has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more channels.
- the substrate comprises a microfluidic slide or microfluidic channel.
- the device is a microfluidic cell tethering slide that can be used to image live cells.
- the device enables tethering of non-adherent cells to allow high-resolution imaging by confocal microscopy of microtentacles and measurement of time-dependent drug responses through media exchange.
- the substrate is made of plastic or glass.
- the device is a microfluidic slide having six channels. Shown in FIG. 17B is a microfluidic slide having six channels in accordance with some embodiments of the invention.
- the substrate of the device comprises 2-10 alternating layers of PMA/PAAm and comprises DOTAP as the tether.
- PEM films composed of PMA/PAAm can be deposited within the microfluidic channels of the device.
- the device is a microscope slide.
- the device is a tissue culture plate or flask, such as a multi-well polystyrene tissue culture plate. The number of wells of the tissue culture plate can vary, and is not limiting. Microscope slides and tissue culture plates and flasks which can be adapted and modified for use in the claimed invention are available from commercial sources (e.g., Corning Inc., Sigma Aldrich).
- the invention provides a method for imaging microtentacles on isolated, living, non-adherent primary tumor cells from a cancer subject comprising:
- the invention provides a method for imaging microtentacles on isolated, living primary tumor cells from a cancer subject, comprising:
- the invention provides a method of identifying a subject with an increased likelihood of having or developing metastatic cancer comprising:
- the invention provides a method of identifying a subject with an increased likelihood of having or developing metastatic cancer comprising:
- the scoring comprises determining whether the one or more cells comprise two or more protrusions longer than the cell radius, wherein the one or more cells comprising two or more protrusions longer than the cell radius indicates an increased likelihood of having or developing metastatic cancer. See, e.g., Balzer, E.M., et al., Oncogene, 2010. 29: 6402-8; Whipple, R.A., et al., Cancer Res, 2010. 70: 8127-37; Balzer, E.M., et al., Breast Cancer Res Treat, 2010. 121 : 65-78.
- the present invention further comprises methods for identifying which candidate drugs should be administered to a cancer patient by assaying whether the drug inhibits, promotes and/or stabilizes the microtentacles.
- drugs which stabilize or promote microtentacle formation should not be administered to patients whereas drugs which inhibit, destabilize or have no effect on microtentacles can be administered.
- These assays can comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the function of one or more microtubule protrusions or one or more components thereof.
- the invention provides a method for determining whether a candidate drug inhibits or promotes microtentacle formation and/or stability on isolated, living, non-adherent primary tumor cells from a cancer subject comprising:
- the invention provides a method for determining whether a candidate drug inhibits or promotes microtentacle formation and/or stability on isolated, living primary tumor cells from a cancer subject comprising:
- the method further comprises administering an effective amount of the candidate drug to the subject to treat the cancer.
- the candidate drug is administered if it inhibits microtentacle formation and/or stability or has no effect on microtentacles.
- the subject is not administered an effective amount of the candidate drug if that drug promotes microtentacle formation and/or stability.
- the term “candidate drug” refers to any molecule that may potentially inhibit or promote microtentacle formation and/or stability.
- the candidate drug is not limiting and can include a protein or fragment thereof, a small molecule, or even a nucleic acid molecule.
- the candidate drug can include approved drugs currently used in cancer therapy, non-approved drugs, investigational compounds, and compounds from libraries which can be screened for activity.
- the candidate drug is selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Ar
- the drug is selected from the group consisting of Paclitaxel, Curcumin, Docetaxel, Ixabepilone, Vinblastine, Colchicine, Y-27632 Fasudil, SU6656 Dasatinib, HDAC inhibitors, ROCK inhibitors, Parthenolide, Costunolide and ML-7 Jazplakinolide.
- U.S. Patent No. 8,193,238 also describes a number of compounds which have potential for inhibition of microtentacles which can be used in the present methods, the disclosure of which is incorporated by reference herein in its entirety.
- the method further comprises imaging one or more living, non-adherent primary tumor cells that have not been treated with the candidate drug and scoring the untreated cells for microtentacles, and comparing the score for untreated cells with the score obtained from step iv) for treated cells.
- the treated and untreated cells are the same cells and the untreated cells are imaged and scored prior to step ii).
- the scoring comprises determining whether the one or more cells comprise two or more protrusions longer than the cell radius. See, e.g., Whipple et al., Experimental Cell Research 313(7): 1326-36 (2007).
- the candidate drug promotes microtentacle formation and/or stability when the one or more cells exhibit a greater number of protrusions longer than the cell radius compared to untreated cells. In some embodiments, the candidate drug inhibits microtentacle formation and/or stability when the one or more cells exhibit a reduced number of protrusions longer than the cell radius compared to untreated cells. In another embodiment, the invention provides a method for determining the stem cell potential of tumor cells from a cancer subject comprising:
- the invention provides a method for determining the stem cell potential of tumor cells from a cancer subject comprising:
- the scoring comprises determining whether the one or more cells comprise two or more protrusions longer than the cell radius, wherein the one or more cells comprising two or more protrusions longer than the cell radius have a greater stem cell potential than cells having fewer than two protrusions longer than the cell radius.
- the number of cells that can be isolated and assayed is not limiting. In some embodiments, fewer than 1000 cells are isolated and analyzed. In some embodiments, fewer than 200 cells are isolated and analyzed. In some embodiments, fewer than 50 cells are isolated and analyzed. In some embodiments, about 12 cells are isolated and analyzed. In some embodiments, at least one cell is isolated and analyzed. The cells can be isolated from surrounding cells and tissue to yield single-cell suspensions using standard techniques.
- Surgical and needle biopsy samples provide a unique resource for early measurements of tumorigenic and metastatic potential in live tumor cells that is amenable to testing individual patient prognosis and drug responses without requiring long-term growth in cell culture.
- Culturing of patient-derived tumor cells can impose selective pressures that yield tumor cell populations which do not reflect the molecular characteristics of the patients' original tumors (DeRose, Y.S., et al., Nat Med, 2011. 17: 1514-20).
- the invention allows a comprehensive mechanical analysis of free-floating (non- adherent), patient-derived tumor cells within hours or even minutes of their removal from the patient during surgery.
- the methods of the invention can be applied on groups of fewer than 200 cells, which would allow their use from even core needle biopsy samples at the time of initial diagnosis.
- the cells are freshly isolated from a solid tumor from the subject and the cells are dissociated and imaged for the micro tentacles.
- the cells are imaged within one week of isolation.
- the cells are imaged within 48 hours of isolation, within 24 hours of isolation, within 12 hours of isolation, within 6 hours of isolation, within 2 hours of isolation and within 1 hour of isolation.
- the cells have not undergone more than three doublings, more than two doublings, or more than a single doubling since isolation from the solid tumor.
- the imaging the one or more living, non-adherent primary tumor cells is not limiting, provided, however, that the imaging allows for the detection of the micro tentacles.
- the cells are imaged using microscopy.
- the cells are imaged by confocal microscopy.
- the cells are imaged by flow cytometry image stream analysis (see, e.g., Imagestream by Amnis (Seattle, WA)).
- one or more membrane markers are labeled to facilitate visualization of the plasma membrane and the microtentacles.
- the plasma membrane is labeled with a fluorescent dye to visualize the microtentacles.
- a lipophilic membrane dye e.g., CellMask, 1 : 10,000, Invitrogen
- a lipophilic membrane dye can be added to the cells to facilitate membrane and microtentacle visualization of live cells. See, e.g., Charpentier et al., Cancer Research 15;74(4): 1250-60 (2014).
- one or more other methods for surface labeling are employed, e.g., WGA (Balzer, E.M., et al., Breast Cancer Res Treat, 2010.
- Tumor cells that can be analyzed by the methods of the invention can be from any type of tumor in which microtentacle formation is involved in metastasis, for example, breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, brain cancer, liver cancer, testicular cancer, thyroid cancer, skin cancer, colon cancer, ovarian cancer, cervical cancer, and uterine cancer.
- the tumor cells are tethered to a substrate during imaging of the cell and microtentacles.
- the nature of the tethering of the cells to a substrate is not limiting provided that the cells maintain their free-floating, nonadherent character, and are able to form microtentacles.
- the substrate is coated with one or more layers of one or more materials that substantially inhibit the cells from adhering to the substrate.
- the substrate is coated with one or more polyelectrolytes.
- the substrate is coated with polyelectrolyte multilayer films (PEMs) to coat the imaging surface to both prevent the formation of protein-based attachments and reduce cell displacement.
- PEMs polyelectrolyte multilayer films
- the PEMs are exceptionally thin (10-100 nm) and can incorporate tethering molecules in their top surface, such as DNA oligos or lipids that can interact with cell membranes and hold free-floating cells without allowing cell spreading.
- microcontact printing can be implemented to allow patterning of these attachment points. Tethered cells maintain the McTN dynamics and drug responses of free-floating cells, but can be held in an arrayed pattern for staining and high-resolution imaging.
- the PEMs are prepared by adsorption of alternating layers of polycationic and polyanionic solutions to the substrate that assemble through electrostatic or hydrogen bonding (see Fig. 17A).
- the films are ultrathin (10-lOOnm), optically clear, and use a simple all-aqueous approach based on sequential exposure of a target surface or substrate to the polyelectrolyte solutions.
- the PEMs are created on microfluidic slides or microfluidic channels (see Fig. 17B).
- the PEMs are formed from a combination of polymethacrylic acid (PMA) and polyacrylamide (PAAm).
- the PEMs comprise about 2-10 layers.
- PEMs are described in, e.g., Jewell et al., Advanced drug delivery reviews 60: 979-999 (2008); Jewell et al., Biomacromolecules 7: 2483- 2491 (2006); jewell et al., Journal of Controlled Release 106: 214-223 (2005)).
- the coated substrate is further coated with a tethering substance, which will adhere to the substrate and is capable of tethering the cell to the substrate and holding it in a substantially fixed position for imaging analysis.
- the tether is not limiting and can include any substance, such as proteins, lipids, nucleic acids, carbohydrates, aptamers, etc.
- the tether is a lipid.
- the tether is a charged lipid.
- the tether is a glycerophospholipid.
- the tether is selected from the group consisting of dotap (l,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt)), 18:0-LysoPG and 15:0(3)-16: 1-CA.
- the cells are imaged while tethered to a microfluidic slide.
- the microfluidic slide comprises a polyelectrolyte multilayer comprising a combination of polymethacrylic acid (PMA) and polyacrylamide (PAAm), and dotap as the tether.
- the microfluidic slide can accommodate a sample volume of as little as 30uL.
- McTNs on the surface of free-floating tumor cells serve as an indicator of their reattachment efficiency during experimental metastasis in vivo (Balzer, E.M., et al., Oncogene, 2010. 29: 6402-8; Matrone, M.A., et al., Oncogene, 2010. 29: 3217-27).
- McTNs are a marker of increased stem cell characteristics (Fig. 8).
- Live-cell confocal microscopy can be used to examine McTN extension and dynamics in freshly-isolated tumor cells from cancer patients (such as those having breast cancer, prostate cancer, colon cancer, and other cancers as described herein) and relate McTN incidence and drug response to the tumorigenic and metastatic properties of parallel patient-derived tumorgrafts.
- Mechanical properties of the fresh tumor cells, tumorgraft-derived cells and fresh-frozen tissue can be compared to determine the durability of mechanical properties with different tumor banking techniques.
- stromal matrix is digested with collagenase/hyaluronidase (12h, 37°C), and epithelial cell organoids are isolated from residual fat cells and lymphocytes by centrifugation (530xg, 5 min.) (DeRose, Y.S., et al., Curr Protoc Pharmacol, 2013. Chapter 14: Unitl4 23).
- Organoid pellets are washed with growth media (Hyclone DMEM/F12+HEPES , FBS, BSA, insulin, hydrocortisone) and subjected to cycles of centrifugation until the purity of epithelial organoids is confirmed (DeRose, Y.S., et al., Curr Protoc Pharmacol, 2013. Chapter 14: Unit 14 23).
- Organoids are dissociated with Trypsin/EDTA to yield a single-cell suspension of epithelial tumor cells. Half of this suspension is labeled with anti-CD45 (Alexa 648, Genetix) to exclude lymphocytes and anti-EpCAM (Alexa488, Miltenyi) to identify tumor cells.
- CellMask lipophilic membrane dye (1 : 10,000, Invitrogen) is added and live cells imaged on an Olympus FV-1000 confocal microscope (Fig. 9) with an incubated stage enclosure (Ibidi). Random fields totaling over 200 cells are blindly scored to determine the percentage of CD45-/EpCAM+ cells displaying two or more McTNs, according to our published methods (Balzer, E.M., et al., Oncogene, 2010. 29: 6402-8; Whipple, R.A., et al., Cancer Res, 2010. 70: 8127-37; Balzer, E.M., et al., Breast Cancer Res Treat, 2010. 121 : 65-78; Vitolo, M.I., et al., Oncogene, 2012).
- McTNs are observed in live cells from patients as well as fixed cells that are purified by binding to anti-EpCAM antibodies (Fig. 9). Moreover, McTNs are enriched in the tumor cell subpopulation with increased cancer stem cell characteristics (Fig. 8).
- the CellMask method is currently the most effective for imaging live cells (Vitolo, M.I., et al., Oncogene, 2012), but numerous other methods for surface labeling WGA (Balzer, E.M., et al., Breast Cancer Res Treat, 2010. 121 : 65-78), GFP-membrane (Whipple, R.A., et al., Cancer Res, 2010. 70: 8127-37)) or microtubule visualization of McTNs (Balzer, E.M., et al., Breast Cancer Res Treat, 2010. 121 : 65-78) can be used as CellMask alternatives.
- McTN counts are a possible indicator of in vivo metastatic potential. This was first tested in cultured breast tumor cell lines (Whipple et al., Cancer Research 2008). In subsequent publications, two different genetic models were used to demonstrate that direct induction of McTNs through microtubule stabilization (Matrone et al, Oncogene. 2010 Jun 3;29(22):3217-27. doi: 10.1038/onc.2010.68. Epub 2010 Mar 15) or actin disruption (Balzer et al., Oncogene. 2010 Dec 2;29(48):6402-8. doi: 10.1038/onc.2010.360.
- McTNs are detectable in fresh patient tumor cells, within hours of when the patient cells are removed during surgery.
- patient cells have been propagated as PDX to retain the important connections to the original patient data (growth, metastasis, etc.).
- Table 1 shows the lowest McTN frequencies are found in HCI-003 (ER+/PR+) and the non-metastatic PDX line (HCI-004). These patient profiles have better overall prognosis. The highest McTN counts are also found in those PDX that grow more rapidly (HCI-OOl , HCI-002), indicating a possible connection with increased stem cell characteristics in cells producing McTNs (Charpentier et al., Cancer Research, 2014). The increased McTN metrics that are enabled by the present invention through image analysis hold the potential to reveal more predictors of clinical outcome (McTN length, # per cell, curvature, motion, etc.).
- the present invention enables the rapid testing of patient tumor cells for responses to cancer drugs without requiring long-term growth.
- the present invention therefore provides a method to determine how an individual patient's tumor cells respond to different cancer treatments, enabling a rapid and functional personalized medicine profile.
- the demonstration of McTNs in PDX cells allows application of these drug testing methods to freshly isolated patient cells, thereby allowing rapid tailoring of a patient's treatment regimen in accordance with the patient's microtentacle drug response profile.
- Patient-derived cells are treated with either vehicle (0. 1 % DMSO) or each of the drugs in Table 2 at the indicated concentration for 30 minutes and then analyzed for McTN incidence.
- the weakly metastatic LnCAP cell lines which was derived from lymph node metastases demonstrates an exceptionally smooth surface, either with confocal or DIC microscopy.
- the more metastatic DU145 (brain met) or PC3 (bone met) cell lines have higher McTN incidences.
- Automated McTN analysis shows that McTN length is generally shorter in the prostate tumor cell lines compared to the HCI-001 breast tumorgraft line (compare Figs. 12 and 13). While there is far less consensus in the prostate cancer field for effective markers of stem cell characteristics, numerous published measures show that LnCAP cells have lower stem cell markers than the DU145 and PC3 cells (Wang et al., International journal of biological sciences.
- McTNs are also observed in other types of cancer cells, such as colon cancer cells and B-cell lymphoma cells (for example, in HCT-116 colon cancer cell lines and Jurkat B-cell lymphoma cell lines). McTNs are likely to be found on many different types of tumor cells with metastatic potential; accordingly, the present methods can be applied to any cancer in which tumor cells display micro tentacles.
- Microtentacles are plasma membrane extensions that occur only when tumor cells are unable to form protein-based attachments (such as when they are in the bloodstream during metastasis). McTNs enable circulating tumor cells to reattach in distant tissues during metastasis and are associated with more aggressive cancer phenotypes and stem cell characteristics.
- non-adherent cells can be challenging to image with advanced microscopy, since they continuously drift through the field of view. Attempting to exchange the surrounding media can displace nonadherent cells, greatly increasing the challenge of adding dyes or drugs to non-adherent cells.
- the current example uses polyelectrolyte multilayer films to coat the imaging surface to both prevent the formation of protein-based attachments and reduce cell displacement. This technology enables tethering of nonadherent tumor cells to allow high-resolution imaging of microtentacles and measurement of time-dependent drug responses through media exchange.
- MRI/PET-CT magnetic resonance imaging
- doctors are currently unable to accurately follow early metastasis.
- imaging limitations make it very difficult to determine by imaging methods (such as MRI) if a tumor is shrinking because the tumor is dying or because it is scattering.
- CTCs circulating tumor cells
- the device uses either of two parallel PEM-based approaches (DNA or lipid) to tether tumor cells to an imaging surface while maintaining free- floating tumor cell behavior.
- the most effective strategy can be prioritized for the analysis of live tumor cells from 40 breast cancer patients. Parallel samples from these patients are transplanted orthotopically into immunodeficient mice. This tumorgraft approach has been demonstrated to recapitulate the metastatic behavior of the patient's original tumor far more faithfully than any tissue culture model. McTN characteristics (frequency, length, number per cell) and drug responses in individual patients can be compared to the molecular characteristics (ER/PR/HER2) of the original patient's tumor, as well as eventual growth and metastasis in the tumorgraft model.
- ER/PR/HER2 molecular characteristics
- Multilayered polyelectrolyte assemblies as platforms for the delivery of DNA and other nucleic acid-based therapeutics.
- Advanced drug delivery reviews 60: 979-999 we will coat microfluidic slides with a PEM that prevents tumor cell attachment to enable the study of free-floating cell behavior.
- PEM a PEM that prevents tumor cell attachment to enable the study of free-floating cell behavior.
- an integrated DNA oligo to tether the tumor cell membrane to a PEM film
- yielden NS Todhunter ME, Jee NY, Liu JS, Broaders KE, Gartner ZJ (2012). Chemically programmed cell adhesion with membrane-anchored oligonucleotides. Journal of the American Chemical Society 134: 765- 768).
- This one-step lipid array will also reduce the number of cells needed from patient samples because the entire volume of the microfluidic channel is 30 ⁇ , allowing viewing of all patient cells in a single channel.
- Initial tests with breast tumor cell lines show that reproducible McTN counts can be obtained from as few as 200 cells per channel, increasing the feasibility that this analysis could even be conducted on core needle biopsy samples, which routinely yield between 2,000 and 10,000 tumor cells (Staler DL, Stewart CC, Stomper PC (2002).
- the most effective PEM microfluidic device (DNA or Lipid) will be prioritized to analyze microtentacles on freshly-derived tumor cells from 40 patients and their responses to 6 common and emerging cytoskeletal cancer drugs that either increase microtubule stabilization and McTNs (Paclitaxel, Ixabepilone) or decrease microtubule stabilization and McTNs (Colchicine, Parthenolide, Vinblastine, Curcumin).
- tumorgraft outcome growth rate, metastasis and organ of colonization
- Such tethered cells can be attached in specific patterns and maintain the dynamic behavior of free-floating tumor cells, including McTN formation.
- solutions can be passed over the cells to allow cell surface staining and the application of drugs without significantly disrupting cell tethering or displacing cells.
- PEMs formed from a combination of polymethacrylic acid (PMA) and polyacrylamide (PAAm) provided a weakly cytophillic surface that supported weak attachment of tumor cells ( ⁇ 5 ) even after 4 hours of incubation (Fig. 19A).
- PMA polymethacrylic acid
- PAAm polyacrylamide
- Ellipsometry showed that the thickness of the PMA/PAAm layer was proportional to the number of layers deposited, ranging from lOnm (1 bilayer) to 90nm (8 bilayers). The optical clarity of the coatings remained close to 100%, irrespective of the number of layers.
- Simple application of the PM A/PA Am film to commercially- available microfluidic chips Fig.
- PAAm is well-studied in terms of its ability to reduce cell attachment without causing cell toxicity and PMA is a key component of polyhydroxyethylmethacrylate (polyHEMA) that has been used widely to prevent cell attachment (Brouquet A, Taleb P, Lot AS, Beauchet A, Julie C, Prevost G et al (2011). A model of primary culture of colorectal cancer and liver metastasis to predict chemosensitivity. The Journal of surgical research 166: 247-254). For these reasons, we expect that this coating will be effective and non- toxic. To control for cytotoxicity, we will culture the 15 cell lines within the microfluidic slides for 3 days and gauge cell death by trypan blue exclusion.
- PMA/PAAm films allow attachment of more than 3 out of the 15 breast tumor cell lines within 240 minutes, or causes toxicity in more than 3 out of 15 cell lines over 3 days, we will test alternative surface compositions or increased numbers of layers.
- PEMs composed of Poly-L-lysine (PLL) and Poly-L-Glutamic acid (PGA) have a very similar ability to prevent MDA-436 and MCF-7 attachment.
- PAAm has an amide side group that has many options for surface modification and cross-linking applications (Lakins JN, Chin AR, Weaver VM (2012). Exploring the link between human embryonic stem cell organization and fate using tension- calibrated extracellular matrix functionalized polyacrylamide gels. Methods in molecular biology 916: 317-350).
- PLL/PGA film can serve as an alternative.
- Ibidi microfluidic slides coated with 2 PMA/PAAm films will be used to test the adhesion of more complex mixtures of patient-derived cells.
- Epithelial cell suspensions are purified by enzymatic digestion (collagenase/hyaluronidase) and differential centrifugation (DeRose YS, Gligorich KM, Wang G, Georgelas A, Bowman P, Courdy SJ et al (2013).
- Patient-derived models of human breast cancer protocols for in vitro and in vivo applications in tumor biology and translational medicine.
- Clin Cancer Res 8: 428-432 can also be used to identify breast tumor cells. Red blood cells are easily identifiable by their small size and concave morphology and fat cells stain very brightly with the lipophilic compound (CellMask) used to visualize microtentacles (Fig.20, left panel), making it relatively simple to exclude these cell types during imaging. c) Integrate a DNA oligo for membrane tethering into the upper PEM surface.
- a short DNA oligo (20nts) will be electrostatically adsorbed to PEMs by incubation (30 ⁇ , 0.2mg/mL) over a PM A/PA Am film with a cationic capping layer of PAAm or the strong polycation, PLL.
- a lipid-coupled complementary DNA strand will be added to suspended MDA-436 and MCF-7 cells34 (Adheren, Inc.).
- Suspensions of 5,000 cells in 30 ⁇ of PBS will then be applied to the channels and hybridized for 30 minutes with the complementary DNA displayed on the PEM-coated channel.
- the ability of cells to remain on the PEM-DNA surface will be tested by repeatedly washing the channel with 50uL of media. The number of cells released will be measured by a Countess automated hemocytometer after each wash.
- DNA-based cell tethering is certainly effective for McTN imaging in cultured cell lines.
- this strategy requires very high concentrations of cells (10 7 cells/ml) for the initial DNA membrane labeling procedure (Selden NS, Todhunter ME, Jee NY, Liu JS, Broaders KE, Gartner ZJ (2012). Chemically programmed cell adhesion with membrane-anchored oligonucleotides. Journal of the American Chemical Society 134: 765- 768), it would be advantageous to develop a more direct strategy for the analysis of clinical samples, where limited numbers of cells are available. In this aim, we will develop methods for the direct conjugation of membrane-tethering lipid to the top layer of the PEM film within the microfluidic channel slide.
- lipids with long, hydrophobic fatty acids is required to associate with the membrane.
- the ideal molecule would also have a charged headgroup to support PEM adsorption.
- the negatively charged phosphate in each glycerophospholipid can be used to interact with the positively charged amide group in the terminal PAAm layer of the PEM film.
- lipids will be prepared for adsorption using the lipid film rehydration technique.
- lipid ratios (1 : 10-10: 1 LysoPG:CA) at total concentrations of 1-10 ⁇ of lipid will be dried under nitrogen, then sonicated (12W, 30sec) in 500 ⁇ of HEPES. The PEM-coated surface or channel will then be exposed to the lipid solution for 5 minutes and washed twice.
- confocal microscopy excludes the out- of-focus light, so increased labeling at the attachment surface will not prevent us from imaging the center plane of the cells with low background.
- a low concentration of the membrane dye CellMaskOrange, 1 :20,000
- confocal imaging allows exclusion of background and high-resolution imaging of the membrane. If the background signal remains too high, there are several other membrane dyes that could be used (Dil, DiO), but might present similar difficulties.
- the ⁇ technique is more complicated than simple solution based adsorption of DNA labels (Aim 1), but the technique is well-established and we do not anticipate significant challenges considering the outstanding instrumentation and expertise available. Suss and EVG proximity/contact aligners, Headway photoresist spinners (for preparation of masters), and SEMs will be the most useful resources. To simplify cell tethering in intact channels, we will design a photoactivatable lipid tether.
- McTNs on the surface of free-floating tumor cells serve as an indicator of their reattachment efficiency during experimental metastasis in vivo (Balzer EM, Whipple RA, Thompson K, Boggs AE, Slovic J, Cho EH et al (2010). c-Src differentially regulates the functions of microtentacles and invadopodia.
- McTNs are a marker of increased tumor stem cell characteristics (Charpentier MS, Whipple RA, Vitolo MI, Boggs AE, Slovic J, Thompson KN et al (2013). Curcumin targets breast cancer stem- like cells with microtentacles that persist in mammospheres and promote attachment. Cancer Res).
- PEM devices we will use the prioritized PEM devices and live-cell confocal microscopy to examine McTN extension and dynamics in freshly-isolated tumor cells from breast cancer patients.
- Tumor cells acquired from residual de-identified patient tissue by the Translational Core lab will be directly transplanted into the cleared mammary fat pad of immunodeficient NOD/SCID mice.
- This system was developed by Dr. Alana Welm at the University of Utah Huntsman Cancer Institute and avoids altering the properties of patient- derived tumor cells through selection with in vitro culture conditions.
- Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes. Nat Med 17: 1514- 1520), Dr.
- Tumor graft lines in-hand (Table 3), that were generated from residual de-identified tissue from breast cancer patients at the UMGCC by the Translational Core lab, or provided by the Huntsman Cancer Institute.
- Table 3 tumorgraft lines in-hand
- Tumor grafts faithfully retain the clinical characteristics of the original patient, tumorgraft results often require many months to develop (DeRose YS, Wang G, Lin YC, Bernard PS, Buys SS, Ebbert MT et al (2011).
- Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.
- the device will be used to analyze fresh patient samples as well as the resulting tumorgrafts from 40 patients during the three years of the project.
- McTN images collected from each cell sample will be analyzed with a custom MatLab algorithm developed for this assay that can automatically identify McTNs but also score metrics that were previously not possible to measure, such as the average number of McTNs per cell (Fig.12).
- Fig.12 the average number of McTNs per cell
- McTN frequency Avg. McTNs per cell, Avg. McTN length
- McTN length the eventual outcome of the tumorgrafts (growth rate, metastatic efficiency, metastatic site)
- growth rate metastatic efficiency, metastatic site
- McTN metrics will also be compared to patient outcome and survival data, but the timeframe of this comparison will exceed the project period and likely take >10 years.
- Novel microfluidic cell tethering device enables microtentacle analysis of free- floating breast tumor cells.
- Cancer metastasis occurs when epithelial tumor cells travel through nonadherent microenvironments, like the bloodstream or lymphatics, to a distant organ (Pantel K and Brakenhoff RH, Nat Rev Cancer 2004; 4(6):448-456; Joosse SA, EMBO Mol Med 2015; 7(1): 1-11).
- the presence of tumor cells in the non-adherent microenvironment of the bloodstream known as circulating tumor cells (CTCs) has been detected in numerous epithelial cancers including breast, prostate, colon, and lung (Joosse SA, EMBO Mol Med 2015; 7(1): 1-11).
- CTCs are an early indicator of clinical spread of disease and their levels correlate with decreased survival of patients with cancer (Cristofanilli M, N Engl J Med 2004; 351(8):781-791 ; Cristofanilli M, J Clin Oncol 2005; 23(7): 1420-1430; Krebs MG, J Clin Oncol 2011 ; 29(12): 1556- 1563).
- monitoring CTCs in patients over time and in response to therapy can provide significant information on tumor response and can even be more sensitive than current imaging methods (de Bono JS, Clin Cancer Res 2008; 14(19):6302-6309; Slade MJ, Br J Cancer 2009; 100(1): 160-166; Pierga JY, Clin Cancer Res 2008; 14(21):7004-7010).
- Some of the techniques currently being employed to analyze CTCs include fluorescence in situ hybridization, sequencing, immunostaining, xenograft transplantation, and RNA or protein-based expression analysis (Pantel K and Speicher MR, Oncogene 2015; Yu M, / Cell Biol 2011 ; 192(3):373-382; Yu M, Science 2014; 345(6193):216-220; Yu M, Science 2013; 339(6119):580-584).
- these methods do not allow for real-time analysis of CTCs in an environment that preserves their free-floating nature.
- Non-adherent breast carcinoma cells for example, produce unique tubulin-based microtentacles (McTNs) that promote tumor cell aggregation (Whipple RA, Exp Cell Res 2007; 313(7): 1326-1336; Yoon JR, Breast Cancer Res Treat 2011 ; 129(3):691-701), reattachment to endothelial layers (Whipple RA, Cancer Res 2010; 70(20):8127-8137; Matrone MA, Cancer Res 2010; 70(20):7737-7741), and retention of circulating tumor cells in the lungs of mice (Balzer EM, Oncogene 2010; 29(48):6402-6408; Matrone MA, Oncogene 2010; 29(22):3217-3227).
- McTNs tubulin-based microtentacles
- McTNs New enabling technologies to image tumor cells, McTNs, and other features in the absence of extracellular matrix (ECM) attachment could vastly improve the understanding of dynamic cell behaviors that occur in the non-adherent microenvironments encountered by CTCs during metastasis.
- ECM extracellular matrix
- These tools could also support opportunities for selective targeting of drugs to McTNs or other structures presented preferentially by CTCs during metastatic spread, as well as help address rising concerns that chemotherapies meant to reduce tumor growth may actually increase metastatic risk (Balzer EM, Breast Cancer Res Treat 2010; 121(l):65-78).
- McTNs form only when protein-based adhesions are absent to create an innovative platform for real-time imaging of the dynamic features of live, nonadherent tumor cells.
- Biomaterials offer many attractive features - stability, biocompatibility, versatile chemistries - for controlling cell adhesion.
- Common approaches include chemically functionalizing surfaces (Kato K, Biotechniques 2003; 35(5): 1014-1018, 1020-1011 ; Lee H, Science 2007; 318(5849):426-430; O'Brien FJ, Biomaterials 2005; 26(4):433-441 ; Okano T, Biomaterials 1995; 16(4):297-303), incorporating cell adhesion peptides (Seeto WJ, Acta Biomaterialia 2013; 9(9):8279-8289; Hersel U, Biomaterials 2003; 24(24):4385-4415; Fierer JO, Proc Natl Acad Sci U S A 2014; 111(13):E1176-1181), and micropatterning using polymer-based soft lithography (Zheng W, Advanced Healthcare Materials 2013; 2(1):95-108) or electrospinning techniques (Min B-M, Biomaterial
- PEMs Polyelectrolyte multilayers
- LbL layer-by-layer
- PEMs have the ability to coat topographically- complex surfaces (e.g., planar, colloidal, microfluidic) and offer programmable surface features and functionalities depending on polymers used to assemble the films.
- PEMs have recently been employed to study CTCs by promoting adhesion to PEMs incorporating cytophilic polymers or cell- adhesive protein domains (Zanina N, Biotechnol Bioproc E 2013; 18(1): 144-154; Best JP, Soft Matter 2013; 9(18):4580- 4584; Reategui E, Adv Mater 2015; 27(9): 1593-1599; Lee H, ACS Nano 2011 ; 5(7):5444-5456). These techniques have demonstrated great potential in capturing and immobilizing CTCs. However, new strategies are needed to study the dynamics of McTNs and other unique metastatic features that form only when CTCs are in nonadherent or circulating environments.
- DOTAP maintained tumor cells significantly more efficiently compared to non-tethered cells seeded on micro-slides coated with PEM only ( Figure 27A and 27B).
- MCF-7 cells exhibited an overall higher cell retention rate over five washes compared to MDA-MB-436.
- the first wash was the most effective for both cell lines with retention of 38% and 80% of MDA-MB-436 and MCF-7 cells, respectively ( Figure 27A and 27B).
- DOTAP was able to tether 15% of MCF-7 cells, while DOPC in contrast, was unable to effectively tether either type of tumor cells.
- McTN frequency of MDA-MB-436 and MCF-7 cells seeded on PEM and PEM-DOTAP coated slides was assessed.
- Blinded McTN counts on cells seeded on PEM-no tether and PEM-DOTAP tether were compared to counts corresponding to previously published methods on low-attach multi-well plates.
- McTN frequency on all three substrates Figure 28 A
- McTNs The major advantage in imaging McTNs over time is being able to study their responses to drugs not only by McTN frequency, but also McTN dynamics.
- McTN dynamics Here we show three dimensional z-stacks of MDA-MB-436 cells treated with the microtubule destabilizing agent, colchicine, and the microtubule stabilizing agent, taxol. Addition of colchicine decreases microtentacles and taxol enhances McTNs ( Figure 29B).
- this study shows that lipid tethering is an effective way to retain the free-floating characteristics of suspended tumor cells and enhances our ability to study their functional properties with high-resolution confocal microscopy.
- One application that is shown in this study is imaging McTN response to drugs in real-time where we can not only assess the effects of drugs on McTN frequency in a population as previously published, but also study other properties at a per-cell level such as McTN dynamics and time-dependent drug responses.
- tumor cells While in the circulation, tumor cells are in a non-adherent microenvironment that is unlike the conditions in a primary tumor or the metastatic site. In these non-adherent conditions tumor cells undergo many biochemical and structural changes that affect their sensitivity to therapies and their overall metastatic efficiency (Matrone MA, Cancer Res 2010; 70(20):7737- 7741 ; Cristofanilli M and Mendelsohn J, Proc Natl Acad Sci U S A 2006; 103(46): 17073-17074).
- the lipid moiety itself also altered cell retention as the DOPC lipid was unable to tether cells more effectively. While the cells are immobilized on the substrate, they still remain free-floating due because PEM prevent cell adhesion.
- lipid tethering technology we were able to perform high-resolution microscopy to image McTNs in real-time. While there was considerable loss of tethered cells with washing, this can be improved in the future through addition of a cross-linking step to strengthen the bond between the lipid and the PEM.
- McTN frequency of cells seeded on PEM-DOTAP did not change compared to cells seeded on PEM-no tether or previously published methods (low-attach plates) (Whipple RA, Cancer Res 2008; 68(14):5678-5688). PEM or DOTAP deposition also did not change cell viability. Without the spatial immobilization of the cell that the tethering offers tumor cells rapidly washed away from the substrate or move out of the field of view during microscopy, which makes real-time imaging of McTNs or other structures in a nonadherent cell practically impossible.
- MDA-MB-436 cells We were further able to leverage our microfluidic tethering technology to characterize the response of McTNs to microtubule-targeting drugs.
- colchicine inhibited McTN formation and taxol enhanced McTN formation of MDA- MB-436 cells (Whipple RA, Exp Cell Res 2007; 313(7): 1326-1336; Balzer EM, Breast Cancer Res Treat 2010; 121(l):65-78).
- CTCs play a critical role in disease prognosis and progression in patients with cancer.
- High numbers of CTCs correlate with increased metastasis and decreased survival of patients with metastatic cancer (Cristofanilli M, N Engl J Med 2004; 351(8):781-791 ; Krebs MG, J Clin Oncol 2011 ; 29(12): 1556-1563; Plaks V, Science 2013; 341(6151): 1186-1188; Yap TA, Clin Cancer Res 2014; 20(10):2553-2568).
- CTC enumeration alone may not be good marker for disease staging and prognosis (Plaks V, Science 2013; 341(6151): 1186-1188). Therefore, improved biologic characterization of CTCs is necessary to better understand their clinical value.
- Patterns of drug sensitivities have been linked to the genetic mutations present in individual CTC samples from breast cancer and lung cancer patients, indicating that a change in tumor genotypes during the course of treatment can lead to drug resistance (Yu M, Science 2014; 345(6193):216-220; Cristofanilli M and Mendelsohn J, Proc Natl Acad Sci U S A 2006; 103(46): 17073-17074; Maheswaran S, N Engl J Med 2008; 359(4):366-377). As shown in this study, tethering tumor cells allows for rapid analysis of their response to specific drugs, which can be done in real-time.
- EMT epithelial-to-mesenchymal transition
- Tethering also allows these studies to be conducted in a manner that more closely recapitulates the free-floating environment found in the circulation.
- CTCs can play a role in informing therapeutic management and disease progression of cancer patients.
- PEM-DOTAP tethers to analyze the characteristics of CTC samples is a new tool to provide better, personalized treatment decisions for patients with cancer.
- MDA-MB-436 and MCF-7 cell lines were purchased from ATCC and cultured with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1 % penicillin-streptomycin solution.
- PMA poly(methacrylic acid)
- PAAm polyacrylamide
- PAH Poly(allylamine hydrochloride)
- DOTAP l,2-dioleoyl-3- trimethylammonium-propane
- DOPC l,2-dioleoyl-OT-glycero-3- phosphocholine
- Cut substrates were cleaned with sequential washing with acetone, ethanol, methanol, and deionized water then charged using an oxygen plasma Jupiter III system (March). These substrates were first immersed in the polycationic solution PAH (0.05M) for 15 minutes then rinsed twice using two separate baths of deionized water at pH 3 to remove any excess polymer. This primer layer was followed by immersion of the substrates into polyanionic PMA (0.01M) for 5 minutes followed by rinsing as above. The substrates were then immersed in a polycationic solution of PAAm (0.01M) for 5 minutes and rinsed. For additional bilayers, the process was repeated without the addition of the primer layer (PAH) until the desired number of bilayers was assembled.
- PAH polycationic solution
- Lipid formulations comprised of 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) or l,2-dioleoyl-3- trimethylammonium-propane (DOTAP) were obtained from Avanti Polar Lipids. These lipids were prepared as 0.01M solutions with pH 3 deionized water and sonicated for 60 minutes in a room temperature water bath. PEMs with a lipid tether were prepared by immersing PEM coated substrates in each lipid solution for 5 minutes followed by two rinsing steps. The final, coated substrates were removed from solution, blown dry with compressed, filtered air, and stored at room temperature prior to characterization.
- DOPC dioleoyl-sn-glycero-3-phosphocholine
- DOTAP l,2-dioleoyl-3- trimethylammonium-propane
- MDA-MB-436 and MCF-7 breast cancer cells were seeded on PEM coated micro-slides (50,000 cells/channel) ranging from 0 to 8 bilayers.
- An Olympus CKX4 microscope was used for all experiments to capture images at 4x magnification.
- Three pictures per channel were taken after cell seeding for each condition to quantify initial cell number (tO). At 6hrs and 24hrs media, was removed from the channel and the channel was washed once before addition of new media. Three images per channel were taken for each condition. The area of the image occupied with cells (as a percent) was quantified using CellProfiler (Broad institute) and the average from three images was calculated. The average percentage for each condition was then normalized to the area occupied at tO.
- Attachment Cell Titer MDA-MB-436 and MCF-7 breast cancer cells were seeded on PEM coated 96-well plates (20,000 cells/well) ranging from 0 to 8 bilayers. At each time point (1, 3, 6, and 24 hours), media was removed from the well and the well was washed once before addition of fresh media. After the 24hr time point an additional wash was done on all wells. Cell number was determined using CellTiter reagent according to manufacturer's instructions. Each time point was normalized to initial cell number from a reading done immediately after cell seeding.
- MDA-MB-436 and MCF-7 breast cancer cells were seeded on PEM coated micro-slides with 4 bilayers (50,000 cells/channel). At 0, 6, and 24 hours Live/Dead (Life Technologies) reagent was added according to manufacturer's instructions.
- Live/Dead (Life Technologies) reagent was added according to manufacturer's instructions.
- live (calcein-AM) green fluorescence, and dead (ethidium homodimer-1) red fluorescence images were taken in triplicate at 4x magnification with an Olympus CKX41 fluorescence microscope. The number of cells in each image was quantified using CellProfiler and percent of live and dead cells were calculated by quantifying green fluorescence positive and red fluorescence positive cells, respectively, and dividing by total number of cells in the phase contrast image.
- MDA-MB-436 and MCF-7 breast cancer cells were plated on 96-well black plates with 4 PEM bilayers (20,000 cells/well). At time 0, 1, 3, 6, and 24 hours Live/Dead reagent was added and read on a plate reader according to manufacturer' s instructions. Relative fluorescence units (RFU) were normalized to time 0.
- REU Relative fluorescence units
- Tethering Washing MDA-MB-436 and MCF-7 cells were seeded on PEM coated micro-slides with 4 bilayers and addition of DOPC or DOTAP (50,000 cells/channel). Cells were incubated for lh to allow for tethering. To quantify initial cell number, three images per channel were taken for each condition at time 0. After lh, existing media was gently removed from the bottom port of each channel and fresh media was added to the top port. Following a wash, three images were taken per channel for each condition using an Olympus CKX41 microscope at 4x magnification. This process was repeated for each wash. The area of the image occupied with cells (as a percent) was quantified using CellProfiler and the average from three images was calculated. The average percentage for each condition was then normalized to the area occupied at time 0.
- DOPC DOTAP
- MDA-MB-436 and MCF-7 cells were seeded on PEM coated micro-slides with 4 bilayers and addition of DOPC or DOTAP (50,000 cells/channel). Cells were incubated for lh to allow for tethering. After lh, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to the top port. This wash was to ensure only tethered cells were analyzed. At 0 and 6 hours after washing, Live/Dead reagent was added according to manufacturer's instructions. Corresponding phase contrast, live (calcein-AM) green fluorescence, and dead (ethidium homodimer-1) red fluorescence images were taken in triplicate.
- MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PEM coated micro- slides with 4 PEM bilayers, PEM coated micro-slides with 4 bilayers and the addition of DOTAP, or a low attach 24-well plate (50,000 cells/channel). Cells were incubated for lh to allow for tethering.
- McTNs were scored blindly in a population of 100 cells/well as previously described. Representative images were taken at 40x magnification with an Olympus CKX41 fluorescence microscope.
- MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PEM coated micro-slides with 4 bilayers and PEM coated micro-slides with 4 bilayers and the addition of DOTAP (50,000 cells/channel). Cells were incubated for lh to allow for tethering. After lh, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to top port. This wash was to ensure only tethered cells were analyzed. After this wash, CellMask orange cell membrane dye was added to each channel to a final concentration of 1 : 10,000.
- McTN imaging was done on an Olympus FV100 confocal laser scanning microscope at 60x magnification. Five luM slices and 20 frames at a 10 sec frame rate were taken for at least five image sets for each condition. Colchicine was added to each channel at a final concentration of 5uM and cells were imaged after 15 minutes. Taxol was added to each channel at final concentration of ⁇ g/ml and imaged after 120 minutes. Cells were again imaged with five ⁇ slices and 20 frames at a 10 second frame rate.
- the new dynamic measurements also show that average McTNs/cell, average McTN length and the range of McTN lengths/cell change rapidly in vehicle-treated control cells. By comparison, the variance of all three measures is reduced in Taxol-treated cells, reflecting the effect of stabilizing microtubules. Individual McTN tips can also be followed with MATLAB particle tracking to quantitate McTN dynamics (Fig. 37).
- Reactive amine groups in the PAAm allow the DOTAP tether to be chemically cross-linked to the PEM with formaldehyde, which dramatically improves retention of tethered tumor cells during multiple washes (Fig. 38B).
- This crosslinking approach enables us to conduct improved imaging, time-dependent drug treatments without causing cell drift, but also illustrates that photo-crosslinking could be used to develop patterned arrays to facilitate tumor cell mechanical measurements and cell fate studies.
- Tethering arrays by photo-crosslinling the tether to the substrate
- the lipid tether is photo-crosslinked directly into a PEM coating to enable a one-step tethering of non-adherent tumor cells on patterned surfaces that allow rapid preparation of microfluidic PEM capture devices for high- throughput clinical use.
- Photo-crosslinking will allow patterns of lipid tethers to be generated on PEM coatings within intact microfluidic channels.
- An aryl azide group is used that can be activated with 380 nm light to form covalent crosslinks with the PEM.
- Fig.39 A photolithography mask is prepared in the Fablab and the Suss mask aligner is used - which is able to pattern features >1 ⁇ - to directly illuminate PEM coated Ibidi microchannels with 7 ⁇ islands and 40 ⁇ center spacing.
- a photocrosslinkable lipid (Fig.39) can be used in this experiment, which can be obtained by Avanti Polar Lipids, Inc. This approach will eliminate the need for microcontact printing.
- patterned arrays The rationale for developing patterned arrays is so that cells can be held close together for rapid imaging of groups (Fig. 40A), but also stay far enough apart so that clustering is minimized (Fig. 40A). Patterned arrays will also enable us to more accurately compare McTN behavior and mechanical measurements with micropipette aspiration and AFM conducted at the same coordinates (Fig.40B). Engineered arrays could also probe questions like the spacing that can promote cell-cell attachment in the presence and absence of drugs (Fig.40C). Finally, the technique can be used to answer the question of whether increases in stem cell characteristics caused by drug treatments result from the selection of cancer stem cells (by killing non-stem cells), or if the mechanical changes imposed by cytoskeletal drugs can actually induce stem cell conversion.
- the invention's tethered cell arrays will enable us to distinguish the effects of stem cell conversion and selection by allowing cell fate to be tracked efficiently as tumor cells grow into stem cell spheres (Fig.40D).
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