MARKERS TO IDENTIFY PRIMARY CELLS FROM TUMOR BIOPSIES

A process that utilizes a panel of primary monoclonal antibodies (mAbs) specific for cell markers that are directly conjugated to fluorophores (Alexa Fluor dyes and Quantum Dots). The mAb-fluorophore conjugates are used to interrogate the presence or absence and relative level of expression of each of the cell markers using laser scanning confocal microscopy. Complex tissues contain various cellular subsets each of which contribute in different ways to the biological behavior and each of which has, if selected well, a unique pattern of cell marker molecules capable of being identified by monoclonal antibodies and therefore provide a specific phenotype. The cell markers may be cell surface or intracellular in location. The expression patterns of heterogeneous mixtures of cells are detected by the mAb-fluorophore conjugates, and are used to decipher the identity of the cells based on their expression or lack thereof, of the cell markers.

It should be noted that the figures are not necessarily drawn to scale and that elements of similar structures or functions are generally represented by like reference numerals for illustrative purposes throughout the figures. It also should be noted that the figures are only intended to facilitate the description of the various embodiments described herein. The figures do not necessarily describe every aspect of the teachings disclosed herein and do not limit the scope of the claims.

DETAILED DESCRIPTION

The systems and methods described herein are directed to a process that utilizes a panel of primary monoclonal antibodies (mAbs) specific for cell markers that are directly conjugated to fluorophores (Alexa Fluor dyes and Quantum Dots). The mAb-fluorophore conjugates are used to interrogate the presence or absence and relative level of expression of each of the cell markers using laser scanning confocal microscopy. Complex tissues contain various cellular subsets each of which contribute in different ways to the biological behavior and each of which has, if selected well, a unique pattern of cell marker molecules capable of being identified by monoclonal antibodies and therefore provide a specific phenotype. The cell markers may be cell surface or intracellular in location. However, the technology to identify, enumerate, isolate, recover and analyze, with minimal perturbation, uniquely identified adherent cells from one or more cellular subsets out of a heterogeneous mixture or complex tissue, does not currently exist. The expression patterns of heterogeneous mixtures of cells are detected by the mAb-fluorophore conjugates, and are used to decipher the identity of the cells based on their expression or lack thereof, of the cell markers. By using predetermined combinations of mAb-fluorophores, it is possible to identify and differentiate between a variety of cell subsets that comprise complex tissues, for example, neoplastic tumors. For exemplary purposes only, the present disclosure focuses on human breast tumors and cellular subsets, including putative cancer stem cells, endothelial progenitor cells, myoepithelial cells, epithelial tumor cells, among others.

The embodiments described herein have the ability to multiplex the detection of several cell surface markers within a given sample because the emission spectra of the selected fluorophores do not significantly overlap with one another, or are excited with different excitation lasers such that their emissions do not interfere with one another. This ability, along with the use of the devised panel of cell surface markers, enables the user to identify several cell populations within a single sample without the need for emission fingerprinting. This approach has yielded an innovative methodology that 1) permits simultaneous enumeration of various 3 cellular elements present within a complex adherent cell sample, 2) provides the opportunity to assess the selected molecular profiles of individual cells from these various cellular compartments, 3) can be available to patients at the time of diagnosis vs. after tumor resection, 4) overcomes limitations to existing technologies, such as laser micro-dissection, and 5) is designed for high throughput analyses.

The embodiments described herein are directed to the development of a multicolor immunofluorescent imaging strategy that enables the user to uniquely identify and enumerate primary human cells with defined phenotypes from complex tumor samples. Beyond providing a novel tool for addressing fundamental biological questions in complex tissues or tumors, the embodiments described herein are positioned to be able to direct individualized treatment strategies for cancer patients at the time of diagnosis, with rapid turn around time, and little to any additional risk. One can also easily envision this platform being applied to the evaluation of other non-neoplastic complex tissues either at risk for or experiencing pathophysiologic processes. The embodiments described herein provide a combination of commercially available fluorophores that would not require emission fingerprinting in order to image multiple distinct fluorophores in a single sample.

TABLE 2Control cell lines used to develop the breasttumor cell subset identification strategy.ESACD44CD10CD24CD133CD309CD34MCF-7++−+*+/−−+HUVECs−++−−+−D283-Med−+−−+−−KG-1a−+−−−−+*A small subset of MCF-7 cells have been reported to express CD133 and exhibit cancer stem cell properties.

TABLE 4Control cell lines used to develop the pancreatictumor cell subset identification strategy.ESACD44CD184CD24CD133CD309CD34MCF-7++−+*+/−−−HUVECs−++/−−−+−D283-Med−+−−+−−KG-1a−+−−−−+HeLa−+++−−−

The expression or lack thereof of the cell surface markers shown herein uniquely identify human breast tumor cell subsets of interest (Table 1). A total of 4 cell lines, MCF-7, Human Umbilical Vein Endothelia I Cells (HUVECs), KG-Ia, and D283 Med were selected based on their expression of the desired cell surface markers (Table 2). Additionally, the same rationale is applied for the identification of human pancreatic tumor cell subsets (Table 3), and its according control cell lines, MCF-7, HUVEC s, D283 Med, KG-Ia, and HeLa (Table 4). The cell lines collectively express the panel of surface markers and were used as controls to demonstrate proof of principle.

As an example, the panel of mAbs used to pair with the fluorophores has been formulated to identify various cell populations residing within a tumor including: epithelial tumor cells, mammary tumor stem cells, myoepithelial cells, and endothelial progenitor cells. For the individual cell populations the user is interested in identifying, there is no single marker that uniquely identifies one cell type from another, necessitating the need for a panel of surface markers in order to uniquely identify the cell type. There have been reports of cell surface marker panels in order to identify one of the cell types we are interested in identifying, but there are no reports of the multiplexed ability to identify all the individual cell populations within a given sample like the panel of cell surface markers we have devised and no reports of application of these markers to primary biopsy specimens. mAbs specific for each cell surface marker of interest were directly conjugated to Alexa Fluors and Quantum dots. In order to detect the expression of all the markers in a single sample, each fluorophore was paired with a mAb, such that the brightest fluorophores were paired with mAbs specific for lowly expressed surface markers and vice versa (Tables 5 and 6).

The mAb-fluorophore conjugation chemistries are already established in literature. However, the mAb-fluorophore conjugates used in this panel are not readily available for purchase, and so the purified forms of each mAb had to be purchased and individually conjugated to each fluorophore. Covalent fluorophore-conjugated mAbs were utilized to minimize the possibility of cross-reactivity and non-specific binding of secondary reagents within a given sample. The mAbs were conjugated directly to Alexa Fluor dyes (Table 1) and Quantum dots (Table 2) using established crosslinking chemistries.

FIG. 3illustrates an exemplary multicolor imaging strategy, according to one embodiment. The present system provides the ability to multiplex the detection of several cell surface markers within a given sample because the emission spectra of the selected fluorophores do not significantly overlap with one another, or are excited with different excitation lasers such that their emissions do not interfere with one another. This ability, along with the use of the devised panel of cell surface markers, enables the user to identify several cell populations within a single sample without the need for emission fingerprinting. To image the conjugates using laser scanning confocal imaging, the following multicolor imaging strategy was utilized (FIGS. 7,8A-D). The 2 Quantum dots (Qdot 605 and 655) along with Alexa Fluor 405 are all excited706by the same ultraviolet excitation laser (405 m), and so were all grouped into a single imaging track. The remaining Alexa Fluor dyes (488, 546, and 647) are all excited by distinct wavelengths of light, and so each were divided into their own imaging track based on the laser used to excite each dye (488, 561, and 633 nm lasers, respectively). Based on the emission spectra illustrated inFIG. 8A, each fluorophore can be spectrally separated707. The emission spectra of all 3 fluorophores excited by the 405 nrn laser do not overlap with one another, and each Alexa Fluor dye thereafter is excited separately, and so the fluorescence detected from each fluorophore can easily be distinguished from one another.

FIG. 4Aillustrates an exemplary Qdot-mAb conjugation scheme used to couple monoclonal antibodies to respective fluorophores.FIG. 4Billustrates an exemplary Alexa Fluor-mAb conjugation scheme used to couple monoclonal antibodies to respective fluorphores. To interrogate the surface markers using laser scanning confocal microscopy, the flow cytometry validated purified mAbs were conjugated directly to Quantum dots (QD400,FIG. 4A), and AlexaFluor dyes (AF401,FIG. 4B) using established cross-linking chemistries. Covalent fluorophore-conjugated mAbs (primary labeling) vs. the use of secondary fluorophore reagents minimized the possibility of cross-reactivity and non-specific binding of secondary reagents within a given sample. In order to detect the expression of all the markers in a single sample, each fluorophore was paired with a mAb such that the brightest fluorophores were paired with mAbs specific for lowly expressed surface markers and vice versa. The resulting mAb-fluorophores were then used to stain and image each cell line (seeFIG. 5A500). Isotype antibodies were also conjugated to each fluorophore for appropriate negative controls (seeFIG. 5B501).FIG. 5Aillustrates exemplary immunoflourescent detection of single cell surface markers using fluorophore conjugated monoclonal antibodies, according to one embodiment.FIG. 5Billustrates exemplary conjugation of mouse IgG1 and rat IgG2b isotype antibodies to respective dyes as controls, according to one embodiment.

Mixtures of the 3 cell lines were also stained and imaged to demonstrate the identification of each cell population based on their expression pattern of the 6 cell surface markers (seeFIG. 6600).FIG. 6illustrates exemplary multicolor immunoflourescent detection of cell surface markers, according to one embodiment.

FIG. 7illustrates an exemplary cell identification strategy700for use with the present system, according to one embodiment. The cell identification strategy700includes detecting the expression patterns of markers that are expressed on cells to uniquely identify different cell populations within heterogeneous mixtures of cells. Cells are selected based on their expression of desired cell surface markers701. The cell markers may be on the cell surface or intracellular in location. The cell markers are interrogated703using the monoclonal antibodies (mAbs) that are directly conjugated702to fluorophores (the mAb-fluorophores). The mAb-fluorophore conjugates are used to detect the presence or absence and relative level of expression of each of the cell markers using laser scanning confocal microscopy704. By using predetermined combinations of mAb-fluorophores, it is possible to identify and differentiate between a variety of cell subsets that comprise complex tissues, for example neoplastic tumors.

FIGS. 8A-Dillustrate emission spectra resulting from a multicolor imaging strategy of fluorophores, according to one embodiment. The fluorophores were imaged using 4 imaging tracks that were separated by excitation laser (vertical lines at 405, 488, 561, and 633 nm). Excitation spectra (dashed lines) and the according emission spectra (solid lines) are as depicted on the graphs of wave length of light (nm) vs. % fluorescence intensity.

This approach has yielded an innovative methodology that 1) permits simultaneous enumeration of various cellular elements present within a complex adherent cell sample, 2) provides the opportunity to assess the selected molecular profiles of individual cells from these various cellular compartments, 3) can be available to patients at the time of diagnosis vs. after tumor resection, 4) overcomes limitation s to existing technologies, such as laser micro-dissection, and 5) is designed for high throughput analyses.

Markers to identify primary cells from tumor biopsies have been disclosed. It is understood that the embodiments described herein are for the purpose of elucidation and should not be considered limiting the subject matter of the disclosure. Various modifications, uses, substitutions, combinations, improvements, methods of productions without departing from the scope or spirit of the present invention would be evident to a person skilled in the art.