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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0693—Tumour cells; Cancer cells
  • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1323—Adult fibroblasts

Definitions

• the present invention is directed to a method of treating cells by co-culturing them with activated fibroblasts in order to regulate the growth and/or status of the cells.
• Fibroblasts are activated by culturing the cells under conditions that induce the cells to adhere to each other to form multicellular aggregates or spheroids.
• the present invention also provides a device for selecting cells from cell samples, such as bone marrow aspirate, the device comprises said multicellular aggregates.
• fibroblasts are ubiquitous sentinel cells (Mayani et al, 1992; Torok- Storb et al, 1999) that modulate series of developmental and pathologic conditions ranging from cell differentiation and organogenesis to inflammation and cancer (Bhowmick et al, 2004). Being the major stromal cellular constituents, fibroblasts play a dominant role in control over differentiation and proliferation of hematopoietic precursors (Greenberg et al, 1988; Kubota et al, 1988; Wang & Sullivan, 1992).
• fibroblasts support proliferation of both normal and malignant hematopoietic precursors (Rogalsky et al, 1991; Bendall et al, 1994; Buske et al, 1994; Bradstock et al, 1996). They are a rich source of several factors governing hematopoiesis (Silzle et al, 2004; Smith et al, 1997). Proper maturation of leukocytes requires strict control over the prevailing proliferative activity of the immature blasts, and is achievable only by a reciprocal complex interaction with their surrounding mesenchyme (Tavassoli&Friedenstein, 1983; Youn et al, 2000). Leukemic cells have, however, lost their ability to translate these control signals properly, and remain undifferentiated and intensely proliferating (Griffin & L ⁇ wenberg, 1986; D ⁇ hrsen & Hossfeld, 1996).
• HGF/SF hepatocyte growth factor/scatter factor
• HGF/SF is a regulator of hematopoiesis, as well (Kmiecik et al, 1992).
• Figures IA, IB and 1C c-Met expression in leukemia cell lines, and growth characteristics of selected cell lines subjected to nemosis.
• A Expression of the c-Met receptor in leukemia cell lines KG-I, THP-I, U-937, Jurkat, Raji, and K562. Arrow indicates position of the properly processed form of c-Met (145 kDa)
• B Proliferation kinetics of leukemia cell lines KG-I, THP-I, and U-937 with and without stimulation by nemotic fibroblast spheroids in co-culture. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 between treatments at indicated time-points.
• C DNA histogram data and percentage of cells as divided into cell cycle GOGl, G2, and S phases of leukemia cell lines KG-I, THP-I, and U-937 with (closed bars) and without (open bars) stimulation by nemotic fibroblast spheroids in co-culture for 96 hours.
• FIGS. 2 A, 2B and 2C Lentiviral vector transduction of c-Met into THP-I cells.
• A Expression of c-Met in the leukemia cell lines KG-I, THP-I, and U-937 stimulated for 96 hours by nemotic fibroblast spheroids (+) and without stimulation (-).
• B c-Met expression kinetics in GFP-control and c-Met-transduced THP-I cells at indicated time points.
• Figures 3A, 3B, 3C and 3D Adherence, morphology, and chemotactic response of the leukemic cells to nemosis.
• A Percentage of cells from the total cell population adhering to culture dish after co- culture stimulation by nemotic fibroblasts as compared to unstimulated cells of leukemia cell lines KG-I, THP-I, and U-937. *** p ⁇ 0.001 compared to respective control cells.
• B Morphology of adherent cells with or without stimulation by nemotic fibroblast spheroids. Cell elongation and presence of pseudopodia evident in stimulated KG-I and THP-I cells whereas, after stimulation, U-937 cells retain their phenotype.
• ICAM-I intercellular adhesion molecule-1
• cytokine interleukin IL-I, IL-6, IL-8, IL-I l, granulocyte-macrophage colony- stimulating factor, GM-CSF, and leukemia inhibitory factor, LIF
• cytokine interleukin IL-I, IL-6, IL-8, IL-I l, granulocyte-macrophage colony- stimulating factor, GM-CSF, and leukemia inhibitory factor, LIF
• FIGS. 7A and 7B Immunoblot analysis of apoptosis-related and intracellular signaling proteins in leukemia cells.
• A Expression of apoptosis-related molecules in leukemia cell lines KG-I, THP-I, and U- 937 with (+) or without (-) stimulation by nemotic fibroblast spheroids in co-culture for 96 hours.
• nemo sis-responsive cell lines KG-I and THP-I activation-associated cleavage of caspase-3 and -8 is evident. No cleavage products of caspase-9 and reduced expression of the cleaved form of poly(ADP-ribose)polymerase (PARP) are visible.
• Phenotype differences between nemosis-responders and the nemo sis-unresponsive cell line U-937 evident in expression levels of the pro-apoptotic Bax protein.
• Increased dephosphorylation of p38 and ERKl /2 is evident in the nemo sis-responsive cell lines KG-I and THP-I together with increased expression of JAKl and JAK3.
• the nemosis-unresponsive cell line U-937 showed no expressional differences for these proteins.
• Figure 8 Schematic model summarizing effects of nemo sis-derived signals on solid and hematopoietic tumor cells.
• the present invention is based on the discovery that clustered or aggregated fibroblasts or other mesenchymal cells, such as bone marrow mesencymal stem cells, are able to induce a cytostatic, growth inhibitory, and/or differentiating response in primary cells in patient samples and cell lines, i.e. target cells, when such clustered cells or the cytokines and growth factors produced by the aggregates are in close vicinity or in contact with target cells, or when target cells are treated with said compounds deriving from said aggregates. Therefore, the invention provides a method for treating a cell sample, or isolating, and/or enriching cells from a cell sample, said method comprising:
• Step b) may also be performed by treating said cell sample with factor(s) derived from or produced by said multicellular aggregates.
• While co-culturing there preferably is a semi-permeable barrier or membrane between said cell sample and the multicellular aggregates, said barrier permitting exchange of cytokines and growth factors but separating physically said spheroids from said cell sample.
• the expression "separating physically” means that the multicellular aggregates are not in direct contact with the cells in said cell sample.
• the pore size of said semipermeable barrier may preferably be 0.2 to 2 ⁇ m.
• the material for the semi-permeable membrane can be polyethylene terephthalate, polycarbonate, mixed cellulose esters, or teflon.
• said cell sample contains mononuclear cells isolated from bone marrow aspirate(s). More preferably, said cell sample is from patient(s) with malignant disease such as leukemia.
• the term "cell sample” refers herein to a sample containing cultured cells, such as cells of a known cell line, or to a biological sample, such as a patient sample, e.g. a blood sample. Other preferable patient sample is a bone marrow aspirate.
• the above method may also comprise a further step of c) selecting those cells from said celll sample which responded to the co-culturing with said multicellular spheroids.
• the selected cells respond to the co-culturing by chemotactic movement.
• said cells are selected for a therapeutic or diagnostic use.
• Said device comprises a first compartment and a second compartment, said first compartment being arranged within the second compartment, wherein said first compartment comprises multicellular spheroids of fibroblast cells or mesenchymal stem cells in a buffer, said first compartment being separated from the second compartment by a semi-permeable membrane allowing the exchange of buffer, cytokines and growth factors between the compartments; and wherein the second compartment is separated from the sample of cells by a second membrane having a pore size allowing cells to migrate across the membrane.
• the pore size may preferably be 3 to 8 ⁇ m.
• the material for the second membrane may be polyethylene terephthalate, polycarbonate, mixed cellulose esters, or teflon.
• said compartments and the cell sample are surrounded by an outer sealing membrane.
• said semi-permeable membrane does not allow the exchange of cells between said compartments.
• One preferred embodiment of the invention is to use autologous fibroblasts in step a), if the cell sample is a patient sample and the cells in the sample are to be used in a therapy of said patient.
• allogeneic fibroblasts can be used in the invention, since the activated fibroblast cells are preferably not in direct contact with the cell sample.
• Antibodies for immunoblotting were rabbit anti-p38 antibody (Ab) (sc-535, Santa Cruz Biotechnology Inc, Santa Cruz, CA), mouse anti-p-p38 Tyrl82 monoclonal antibody (MAb) (sc-7973), rabbit anti-JNK Ab (CST-0252, Cell Signaling Technology, Danvers, MA), mouse anti-p-JNK Thrl83/Tyrl85 MAb (sc-6254), rabbit anti-ERKl/2 Ab (sc-94), mouse anti-p-ERKl/2 Tyr204 MAb (sc-7383), rabbit anti-Akt Ab (CST-9272), rabbit anti-p-Akt Ser473 Ab (CST-9271), rabbit anti-JAKl Ab (sc-7228), rabbit anti-JAK2 Ab (sc-294), rabbit anti-JAK3 Ab (sc-513), rabbit anti-TYK2 Ab (sc-169), rabbit anti- cleaved caspase-3 Asp 175 Ab (CST-9661), mouse anti-p38
• Antibodies for flow cytometry from the Beckman Coulter Company (Miami, FL) were: anti-CDla-PE (IM1942), anti-CD3-PE (IM1282), anti-CDlO-PE (IM1915), anti- CDl Ia-FITC (IM0860), anti-CD 1 Ib-PE (IM2581), anti-CDl Ic-PE (IM1760), anti-CD13- PE (IM1427), anti-CD 14-FITC (IM0645), anti-CDl 5 -FITC (IM1423), anti-CD 16-FITC (IM0814), anti-CD28-FITC (IM1236), anti-CD33-PC5 (IM2647), anti-CD34-PC5 (IM2648), anti-CD38-FITC (IM0775), anti-CD40-PE (IM1936), anti-CD41-FITC (IM0649), anti-CD45-FITC (IM0782), anti-CD45RA-FITC (IM0584), anti-CD45RO-PE
• HFSF- 132 Cell cultures - Cultures of foreskin-derived human fibroblasts, HFSF- 132, were used from passages 7 to 15 as described (Bizik et al, 2004). KG-I, THP-I, U-937, K562, Jurkat, and Raji were from the American Type Culture Collection (ATCC, Manassas, VA). All cells were cultured in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% fetal bovine serum (Life Technologies), 100 Ag/mL streptomycin, and 100 units/mL penicillin.
• Spheroid formation was initiated as described by Bizik et al, 2004. Briefly, U-bottom 96- well plates (Costar, Cambridge, MA) were treated with 0.8% LE agarose (BioWhittaker, Rockland, ME) prepared in sterile water to form a thin film of a nonadhesive surface. Fibroblasts were detached from culture dishes by trypsin/ EDTA, and a single cell suspension (4x10 4 cells/ ml) was prepared in a complete culture medium. To initiate spheroid formation, 250 ml aliquots were seeded into individual wells and the dishes incubated at +37 0 C in a 5% CO 2 atmosphere.
• the leukemia cells were cultured for various time-periods with 24-hour-preformed fibroblast spheroids at a 1:1 leukemia cells: fibroblast ratio.
• cell numbers were evaluated by cell-counting in B ⁇ rker chambers.
• FACS fluorescence-activated cell sorting
• adherence testing the residual spheroids were removed from co-cultures by gravitational differential sedimentation.
• Morphology of leukemic cells 96 hours after co-culturing was evaluated by phase contrast microscopy.
• the leukemic cells' adherence was estimated after 96 hours of co-culturing with fibroblast spheroids. Thereafter aliquots of cell lines were seeded onto standard cell- culture dishes for 24 hours. The cultures were washed, and adherent cells were harvested by trypsinization, were counted, and the percentage of these adherent cells was calculated.
• Chemotaxis of leukemic cells was performed in agarose-treated 6-well plates as co- cultures of 24-hour-preformed fibroblast spheroids with the na ⁇ ve leukemia cell lines. We calculated with an ocular grid the number of leukemic cells located at a distance from the spheroid double its own diameter, and measured these cells around 15 spheroids per well.
• Immunoblotting - Cell samples were lysed directly in SDS-PAGE sample-loading buffer: 62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 5% ⁇ -mercaptoethanol, and 0.005% bromophenol blue, supplemented with Complete Mini-protease inhibitor mixture tablets (Roche, Mannheim, Germany) and boiled for 5 minutes. Lysates were centrifuged at 14,000 rpm for 15 minutes to sediment particulate-insoluble material. These samples were separated in SDS-PAGE (gradient of polyacrylamide 5-15%, 3.5% stacking gel).
• the proteins were transferred electrophoretically from the gel to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), with transfer efficiency verified by Ponceau-S staining. After blocking of the membrane with 2.5% low-fat dry milk in TBS, 20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20 at pH 7.5, it was incubated with specific primary antibodies, followed by an alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI). Protein bands were visualized according to manufacturer's recommendations.
• Flow cytometry For flow cytometric analysis, the leukemia cells co-cultured for indicated time points and after differential sedimentation to remove spheroids were incubated on ice with antigen-specific antibodies or with isotype-matched antibodies as controls, and fixed in 1% paraformaldehyde. FACS analysis was done by an EPICS ALTRA flow cytometer with the EXPO32 analysis program (both from Beckman Coulter Inc, Fullerton, CA).
• Fibroblast spheroid-conditioned medium was collected at 96 hours after initiation of spheroid formation from the 96-well plates. Concentrations of IL-I ⁇ , IL-6, IL-8, IL-I l, GM-CSF, LIF, oncostatin M, and TNF- ⁇ were quantified by commercial ELISA kits and reagents according to manufacturers' instructions.
• the human IL- l ⁇ , human IL-I l, human LIF, human TNF- ⁇ , and human oncostatin M ELISAs were from R&D Systems (Minneapolis, MN), the human IL-6 and human IL-8 ELISAs were from the Central Laboratory of the Netherlands Red Cross (CLB, Amsterdam).
• Cytokine quantification in the nemotic fibroblast-conditioned medium for human IL-2, IL-4, IL-5, IL-IO, IL- 12, IL- 13, GM-CSF, interferon- ⁇ (IFN- ⁇ ), and TNF- ⁇ was carried out with the Bio-Plex Human Cytokine Thl/Th2 Panel (Bio-Rad Laboratories Inc, Hercules, CA, catalogue number 171- Al 1081), by the Luminex 100 System (Luminex Corporation, Austin, TX).
• Phcnotypic and growth characteristics and cell cycle analysis In analysis of leukemia cell lines for their expression of the HGF/SF receptor c-Met, expression of the properly processed form appeared on U-937, Jurkat, Raji, and K562 cell lines but not on THP-I or
• FIG. 2 A to 2C show the growth responses when these cell lines were subjected to fibroblast nemosis. Attenuation of growth by nemosis treatment was evident at 72 hours of co-incubation for the KG-I cells, but was already evident at 48 hours in the THP-I cell line, demonstrating the more rapid response and reactivity to nemosis of the latter cells.
• Figures 2D to 2E show the leukemic cell lines' cell-cycle phase distribution as evaluated by DNA histograms.
• NSAIDs non-steroidal antiinflammatory drugs
• Cytokine production in ncmotic fibroblasts We previously reported that nemotic fibroblasts are a rich source of the c-Met ligand HGF/SF. Based on our data, and the lack of any effect by NSAIDs, it seemed obvious that the lack of c-Met in the nemosis- responsive leukemia cell lines ruled out any role for HGF/SF in induction of growth arrest. Moreover, the chemotactic response suggests involvement of chemoattractants. We therefore evaluated a pattern of cytokines known to be associated with modulation of chemotaxis and leukemia cell proliferation.
• the cytokines produced most abundantly by the spheroids were IL-6 and IL-8, with fold- inductions (mean production in spheroids) of 3.7 (25.4 ng/ml) and of 8.0 (158.7 ng/ml) as compared to the corresponding monolayer cultures.
• fold- inductions of 18.3 and 3.8 occurred in the production of IL-I ⁇ and LIF.
• Nemotic fibroblasts also produced GM-CSF, which was undetectable from monolayer cultures (Figure 6).
• Apoptosis-related intracellular changes in the leukemia cell lines by nemosis - Inhibition of tumor cell growth is usually accompanied by induction of apoptosis.
• Figure 7 A expression of several apoptosis-associated proteins revealed that the cleaved, active form of the universal apoptosis executor, caspase-3, occurs in response to nemosis only in the nemosis- responsive cell lines. That the unresponsive U-937 showed no effect suggests activation of apoptosis in the nemo sis-responsive cells.
• PARP poly-ADP-ribose- polymerase
• JAK Janus protein tyrosine kinase family
• nemotic fibroblasts are rich producers of cytokines and growth factors such as IL- l ⁇ , IL-6, IL-8, IL-I l, LIF, and GM-CSF, all of which are involved in regulation of hematopoiesis and differentiation (Lotem & Sachs, 2002; Zhu & Emerson, 2002).
• Leukemic cells represent an undifferentiated phenotype of white blood cells, and inducing their differentiation toward a dendritic cell-like type has stimulated therapeutic antileukemic T-cell responses (Charbonnier et al, 1999; Choudhury et al, 1999; Cignetti et al, 1999; Fujii et al, 1999; Claxton et al, 2001; Mohty et al, 2002;
• nemotic fibroblasts can also drive a phenotype-dependent differentiation of leukemia cells in terms of morphological features, functional responses, and surface antigen expression.
• Differentiation of THP-I and KG-I cells in response to nemosis clearly represents an adherent, mature antigen-presenting cell- like type, as further characterized by FACS.
• CD45 -gating CD45 and CD45RA are expressed on all hematopoietic cells except mature red blood cells and their immediate progenitors, with increased levels of these antigens correlating with degree of differentiation (Hermiston et al, 2003).
• CDlIc ⁇ 2 integrin CDlIc
• CDlIc a marker for myeloid dendritic cells (Osugi et al, 2002) associated with differentiation and maturation (Corbi&Lopez-Rodriguez, 1997; Noti&Reinemann, 1995), we found to be linked to the induction of co-stimulatory molecule CD86 in the nemosis-responders.
• CDl Ic acts as an adhesion molecule mediating cell-cell and cell-matrix interactions (Shelley et al, 2002).
• CD86 (B7-2) binds CD28 and CTLA-4 molecules on T-cells mediating co-stimulatory signaling (Collins et al, 2002) to enhance T-cell proliferation, activation, and clonal expansion (Coyle et al, 2001). Stimulation of T-cell CTLA-4 enhances antitumor activity and of CD28 enhances the cytotoxic T-lymphocyte-mediated destruction of tumors (Zheng et al, 1998). Similar to CDl Ic, expression of CD86 on leukemia cells has been associated with a dendritic cell- like phenotype (Re et al, 2002).
• ICAM-I Similar to CDl Ic, ICAM-I also mediates transendothelial migration of leukocytes and, like CD86, ICAM-I binding functions as a co-stimulatory signal for the activation of T cells in antigen presentation (Zuckerman et al, 1998; Grakoui et al, 1999; Hubbard&Rothlein, 2000).
• PARP has several functions, and its increased activity and expression are associated with damage to DNA and DNA repair, with cell proliferation and differentiation, and with regulation of transcription (Ame et al, 2004).
• the role of PARP as a causal effector of apoptosis is equivocal (Leist et al, 1997).
• Caspases 3 and 8 are important regulators of differentiation processes not only in monocytes but also in skeletal muscle cells and osteoblasts (Launay et al, 2005). In hematologic and other types of malignant cells, downregulation of caspase-8 serves as a means to resist apoptosis (Hopkins-Donaldson et al, 2003, Yang et al, 2003). Upregulation of caspase-8, as shown here by the action of fibroblast nemosis on leukemic cells, thus suggests that these nemosis-responsive cells act in an opposite-and in a more benign- manner.
• p38 has been shown to prevent Jurkat T-cell apoptosis (Nemoto et al, 1998), and to inhibit all-trans-retinoid acid- induced differentiation of one acute pro-myelocytic cell line (Alsayed et al, 2001).
• nemosis may influence responses of the immune system to malignancy (Figure 8). Differentiation of leukemic cells into the dendritic cell lineage can stimulate anti- leukemic actions of T-cells (Charbonnier et al, 1999; Choudhury et al, 1999; Cignetti et al, 2004); such differentiation can be suggested as immunotherapy (Claxton et al, 2001; Mohty et al, 2002; Buchler et al, 2003). Our results present the first in vitro evidence that homotypic stromal cell-cell interactions leading to nemosis can provide sufficient signaling to modulate and restrain neoplastic growth.
• Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses. Blood 93, 780-786.
• CD34(+) acute myeloid and lymphoid leukemic blasts can be induced to differentiate into dendritic cells. Blood 94, 2048-2055.
• CDl Ic integrin gene promoter activity during myeloid differentiation Leuk. Lymphoma. 25, 415-425.
• Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells. Biochem. Biophys. Res. Commun. 174, 831-838.
• Hepatocyte growth factor is a synergistic factor for the growth of hematopoietic progenitor cells. Blood 80, 2454-2457.
• Bone marrow fibroblasts-conditioned medium regulates the proliferation of leukemic cells. Tokai J. Exp. Clin. Med. 13, 45-51.
• IMM-I intercellular adhesion molecule-1
• LFA-I lymphocyte function-associated antigen 1
• Leukemic dendritic cells potential for therapy and insights towards immune escape by leukemic blasts. Leukemia 16, 2197-2204.
• JAK3 a novel JAK kinase associated with terminal differentiation of hematopoietic cells. Oncogene 9, 2415-2423.

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