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Breast cancer cells expressing stem cell markers

Cancer Immunol Immunother (2009) 58:1185–1194

DOI 10.1007/s00262-008-0623-1

ORIGINAL ARTICLE

Breast cancer cells expressing stem cell markers CD44+ CD24lo are eliminated by Numb-1 peptide-activated T cells

Takashi Mine · Satoko Matsueda · Yufeng Li · Hiroshi Tokumitsu · Hui Gao ·

Cristopher Danes · Kwong-Kwok Wong · Xinhui Wang · Soldano Ferrone ·

Constantin G. Ioannides

Received: 22 May 2008 / Accepted: 29 October 2008 / Published online: 2 December 2008

? Springer-Verlag 2008

Abstract Cancer stem cells (CSC) are resistant to chemo-and radiotherapy. To eliminate cells with phenotypic mark-ers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcita-bine (GEM), paclitaxel (PTX) and 5-X uorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide acti-vated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial speci W c antigen+ (ESA) CD44hi CD24lo, CD44hi CD133+, and CD133+ CD24lo] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEM Res MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEM Sens MCF7. The levels of Numb, and Numb-L-[P]-Ser265 were similar in GEM Res and GEM Sens MCF7 cells. Only the levels of Numb-L (long)-Ser295 decreased slightly. This W nding suggests that Notch-1 cleavage to TMIC is inhibited in GEM Res MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112–2120) and Numb-1 (87–95) eliminated NICD positive, CD24hi CD24lo MCF7 cells. It is likely that the immunogenic Numb-1

Electronic supplementary material The online version of this article (doi:10.1007/s00262-008-0623-1) contains supplementary material, which is available to authorized users.

T. Mine · S. Matsueda · Y. Li · K.-K. Wong · C. G. Ioannides Department of Gynecologic Oncology,

The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

T. Mine

Departments of Immunology and Surgery,

Kurume University School of Medicine, Kurume, Japan

H. Tokumitsu

Department of Cell Signaling, Faculty of Medicine, Kagawa University, Kagawa, Japan

H. Gao

Department of Molecular Pathology,

The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

C. Danes

Department of Molecular and Cellular Oncology,

The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA Y. Li

Department of Melanoma Medical Oncology,

The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

X. Wang · S. Ferrone

Departments of Surgery, Immunology and Pathology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15123, USA

C. G. Ioannides

Department of Immunology,

The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

T. Mine (&)

Multidisciplinary Treatment Center,

Kurume University School of Medicine,

67 Asahi-machi, Kurume 830-0011, Japan

e-mail: mine@med.kurume-u.ac.jp

peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wort-mannin and MAPK-inhibitors decreased peptide presenta-tion. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments.

Keywords Drug resistance · Breast/ovarian ·

Cancer stem cell · Notch · Numb · Peptide

Introduction

Renewal of embryonic (ESC) and adult stem cells (ASC) is regulated by signals from the surrounding environment. It is possible that cancer stem cells (CSC) originate from normal-ASC, or their intermediate progenitors (IP) which accumulate oncogenic mutations. More recently, they were shown to originate from ASC which activate embryonal di V erentiation programs. Regardless of their origin CSC renew after activation by Notch and ampli W cation by Hedgehog and -catenin (Wnt)-signals of Notch signals [2, 20, 23].

Breast cancer cells with high levels of CD44 (CD44hi) and absent or low CD24 (CD24neg/lo) expression have func-tional characteristics of CSC. Brain, colon and prostate cancer cells with CSC-characteristics express CD133 (prominin-1) [1, 3, 10, 28, 31].

The levels of CD44 (an adhesion molecule which binds to hyaluronate) directly correlate with metastasis in breast cancer. The levels of CD24 (an adhesion molecule that binds P-selectin) inversely correlate with survival of ovar-ian cancer patients. Studies in human mammary cells sug-gest that the most primitive mammary cells do not express estrogen-receptors, but are capable to di V erentiate to estro-gen positive, luminal epithelial cells [6, 18, 24, 33].

CSC are more resistant to chemo- and radiotherapy than non-CSC [17, 25]. Histone-deacetylase (HDAC) and poly-A-ribose polymerase (PARP)-inhibitors are weak anti-CSC e V ectors. To avoid toxicity, it was proposed to lower doses of standard chemotherapy when used together with HDAC/ PARP inhibitors [12, 26, 27].

A novel approach to cancer therapy is to eliminate CSC with immune e V ectors. We hypothesized that we can elimi-nate CSC by targeting peptides from the antagonistic Notch and Numb proteins with cytotoxic T lymphocytes (CTL). When CSC renew or become quiescent Notch and Numb induce degradation of each other, respectively. Notch and Numb are degraded to peptides by the proteasome. Peptides from NICD and Numb proteins are presented by HLA-class I molecules of cancer cells to T cells.

Hetero-dimeric Notch (hNotch) consists of an extracel-lular domain named NECD and a trans-membrane and intracellular domain, named NICD. NICD and NECD are linked by disul W de bonds. hNotch, located trans-membrane, is activated by its ligands. Human Notch-ligands belong to the Jagged (Jag) and Delta-like (DLL) families. Activation/ inhibition of Notch can proceed in Cis- by Notch ligands expressed on the responding cell or in Trans-from Notch-ligand expressed by neighboring cells. Notch signals are transduced i by NICD, which activates gene transcription [14].

Numb has four isoforms. They form two groups Numb-Long (Numb-L) and Numb-Short (Numb-S) which di V er in length by 5kDa. Each group has two iso-forms, which di V er by 1kDa, and are di Y cult to distin-guish. Both Numb-L and Numb-S are present in cancer cells. However, most human cancer studies do not resolve Numb-S from Numb-L and Numb-like [19]. Non-phosphorylated Numb is adjacent to membrane. After receiving external signals Numb is [P]-lated at Ser295 and migrates to cytoplasm. In cytoplasm Numb-[P]-Ser295 cannot bind NICD to block cell-cycle activa-tion. [P]-lation of Numb at other/additional sites directs its degradation by proteasome. The cells which degrade most Numb divide symmetrically, while the cells which do not degrade cytoplasmic Numb divide asymmetri-cally [4, 32].

Symmetric division of stem cells which lack Numb, dou-ble the number of “mother” stem cells. Asymmetric divi-sion of cells which contain both Notch and Numb results in one “mother” stem cell and one di V erent cell [daughter/IP-stem cell [4, 32]]. Degradation of NICD by Numb inhibits activation of transcription by NICD and cells become quiescent [9, 11, 19].

The aim of this study was to determine whether: (1) the proportion of cells with CSC-phenotype/putative CSC increases in drug-resistant cancer lines, and (2) drug-resis-tant cells with CSC-phenotype can be eliminated by Notch-and Numb-peptide activated CTL.

We found higher numbers of CD44hi CD24lo, CD44hi CD133+ and CD24lo CD133+ cells in gemcitabine (GEM Res), paclitaxel (PTX Res) and 5-X uorouracil (5-FU Res) resistant breast and ovarian cancer lines compared with Drug sensitive (Drug Sens) cells. Three-four times more Drug resistant (Drug Res) cells expressed NKG2D receptors than Drug Sens cells. NICD+ and CD44hi CD24lo cells were eliminated by Notch-1 (2112–2120) and Numb-1 (87–95) peptide-activated peripheral blood mononuclear cells (PBMC) and to a lesser extent by interleukin (IL)-2-acti-vated PBMC.

Materials and methods

Cell lines and drugs

MCF7 and SK-OV-3 cell lines were from ATCC (Manassas, VA). Cells were cultured in RPMI 1640 medium with 10% FBS, 100U/L penicillin, and 100 g/mL streptomycin. We used gemcitabine (Eli-Lilly Indianapolis, IN), paclitaxel (Bristol-Myers Squibb, Princeton, NJ), 5-X uorourcil (Sigma Chemical Co. St Louis, MO) and human recombinant DLL4 (rhDLL4, R&D Systems, Inc. Minneapolis, MN).

Antibodies

The mAb to human antigens included anti-ESA (Biomeda, Foster City, CA), anti-CD44 (BD Pharmingen, San Diego, CA), anti-CD44 (BD), anti-CD24 (BD), anti-CD24 (Abcam Inc., Cambridge, MA), anti-MHC class I chain-related gene A (MICA)/MHC class I chain-related gene B (MICB) (R&D Systems, Minneapolis, MN), anti-CD133/2 (Milte-nyi Biotec Inc., Auburn, CA), and anti-Notch-1 (BD). Polyclonal Abs included anti-Notch-1 (Santa Cruz Bio-technology, Inc., Santa Cruz, CA), anti-NICD, anti-Numb, and anti-Bcl-2 mAb (Abcam).

mAb against rat Numb [P]-Ser264 and Numb [P]-Ser283=Human Numb-L-P-Ser265 and 295 were prepared by Dr. Hiroshi Tokumitsu [34, 35]. Numb has many N-terminal splice variants. The position of the corresponding residues to [P]-Ser264 and [P]-Ser283 of rat Numb di V ers among iso-forms. In human Numb-S is [P]-Ser265 and [P]-Ser284 [Numb-3 (NP_003735) and Numb-4 (NP_0010057450)]. Rat [P]-Ser276 is [P]-Ser295 in human Numb-L [Numb-1 (NP_001005743) and Numb-2 (NP_001005744)]. Rat [P]-Ser283 corresponds to Ser295 in human Numb-L. We used the designations [P]-Ser265 and [P]-Ser295 since they are close to the [P]-Ser positions reported in the recent literature.

IC50 of anticancer drugs

The 50% inhibitory concentration (IC50) of GEM, PTX and 5-FU was determined after a 72-h incubation with drugs as described [13].

Flow cytometry

Cells (25–30￡106) were cultured with drugs at 2￡IC50 for 4days followed by drug at 0.5–1.0￡IC50 for the next 3–6days. Surviving cells (2￡105) were incubated W rst with 20 g of human IgG (Sigma) for 1h on ice to inhibit non-speci W c binding of speci W c mAb during staining. Analysis was performed using a Becton Dickinson FACS Calibur with Cell Quest software (BD). Expression of CD24, CD44 and CD133 was quanti W ed in gated ESA+ cells as described [16].

CSC-like and their progenitors were characterized following the geometrical mean (x2) of CD24 in CD44hi cells. CD24negative=MFI, 0–10, CD24lo=MFI, 10–100, CD24hi=MFI, 100–1,000 [16]. ESA+ [(CD44hi and CD24lo/hi), and (CD133+)] cells were quanti W ed using the formula: (% of total ESA+ cells)￡(% of cells positive for both markers)￡(number of cells in the culture).

Activation of GEM Res MCF7 cells

Cells were stimulated for 24h in serum free medium with 62.5ng/mL rhDLL4 then cultured with or without GEM for 7days. GEM Res MCF7 cells were stimulated three times with DLL4 (62.5ng/mL) weakly for 24h.

Sensitivity of GEM Res MCF7 cells to HLA-A2+ PBMC activated by Notch- and Numb-peptides

Notch-1=NICD peptide (2,112–2,120) and Numb-1= Numb-1-PTB domain peptide (87–95) were previously identi W ed by us as immunogenic [15]. Non-adherent PBMC were activated with peptide-pulsed autologous immature monocyte-derived dendritic cells (iDC), or T2 cells as we described [37]. In all activation procedures, we used IL-12 as co-factor followed by IL-2 48h later. Immunoselection

Because autologous T cells to MCF-7 are not available, we used allogeneic immunoselection. GEM Res MCF7 cells (1.0￡105) were co-cultured in six-well plates with 3.0￡105 IL-2-activated, Notch-1-, or Numb-1-peptide-activated PBMC for 5days. IL-2 was not added to cultures for 72h before use. Residual IL-2 was washed out. Surviving MCF7 cells were analyzed by X ow-cytometry.

Western blot

Cell lysates with nuclei removed were prepared from live MCF7, and SK-OV-3 cells. Immunoblotting and quanti W-cation of NECD, NICD, Numb, and Bcl-2 utilizing -actin as a reference was performed in relation to actin in the same sample, as we described [8, 37].

Results

The number of ESA+ [(CD44+ CD24lo), (CD44+ CD133+), and (CD24lo CD133+)] cells increase in drug-resistant MCF7 and SK-OV-3 cells

We quanti W ed the IC50 of GEM, 5-FU and PTX for MCF7 and SKOV-3 cells. The IC50 (GEM) for MCF7 cells was

430 nM and the IC50 (GEM) for SK-OV-3 cells was 16.1nM. The IC50 (5-FU) for MCF7 cells was 1,302nM. The IC50 (5-FU) for SK-OV-3 cells was 3,560nM. MCF7 cells were more sensitive to 5-FU than SK-OV-3 cells, and more resistant to GEM than SK-OV-3 cells. The IC50 (PTX) was similar for both cell lines=4–5nM.

To verify that ESA levels are the same in drug-resis-tant and sensitive cells we determined expression of ESA. The levels of ESA were the same in Drug Res and Drug Sens cells (Fig.1). Drug Res MCF7 cells were luminal cells (Fig.1 and Supplementary Table1). The number of cells with CSC-markers increased W ve and tenfold in MCF7 and SK-OV-3 cells, respectively. 5-FU and PTX selected CSC-like cells with similar e Y ciency with GEM. The number of CD133+, CD133+ CD44hi and CD133+ CD24lo cells increased by 3–5 fold in GEM Res MCF7 cells and GEM Res SK-OV-3 cells compared with the Drug Sens cells (Fig.2d).GEM Res MCF7 cells have lower levels of NECD

than GEM Sens cells

We found di V erences in the expression of antagonistic pro-teins Notch and Numb, between Drug Res and Drug Sens cells. Reducing conditions break the disul W de bond between NECD and TMIC. TMIC is further cleaved between Ala1710 and Val1717 to generate the Notch-extracellular trun-cated domain (NEXT). NEXT is further cleaved at Val1744 to generate free NICD [5, 21, 30].

NECD levels decreased in GEM Res MCF7 cells com-pared with GEM Sens MCF7 cells. Longer ER-precursors of Notch-1 were present in similar amounts in GEM Res and GEM Sens MCF7 cells (Fig.3a). TMIC levels also decreased in GEM Res MCF7 cells. The amount of NICD was low and similar in GEM Res and GEM Sens cells. The decrease in Notch-1 suggested that Notch-1-NECD was degraded and not replaced, since the amount of its ER-precursor was

Fig.1a, b Increase in number of CD44+ CD24lo cells in GEM Res MCF7 and SK-OV-3 cells. MCF7 [MFI (CD24lo)]

cells range 10–100. c CD24lo MCF7 cells (MFI between 5 and 50) increase in number in

5-FU Res and PTX Res MCF7 cells. This W nding was con W rmed in two additional independently performed experiments

Forward scatter

UT GEM Res UT GEM Res

MCF7SK-OV-3

98.2%95.4%

99.7%97.5%

CD24

0.0 1.315.50.3 6.310.20.0 4.695.10.151.546.0

R3R4R5R3R4R5

R3R4R5

MCF7

UT GEM Res

PTX Res5-FU Res

R2R3R4

R2R3R4R2R3R4

R2R3R4

CD24

0.29.739.10.513.525.6

0.922.028.60.924.125.5

higher. This means that less of Notch-1-ER-precursor was cleaved to TMIC (Fig.3a). The levels of TMIC were lower than in control cells. The levels of Numb were slightly lower in GEM Res and untreated MCF7 cells (Fig.3a). The ratio of NECD and TMIC (pre-NICD) to Numb-L and Numb-S decreased (see OD values). Therefore “quiescent”GEM Res MCF7 cells have lower levels of Notch-NECD than “quiescent” GEM Sens MCF7 cells. Control, dividing SK-OV-3 cells synthesized high amounts of Notch and expressed both TMIC (of 100kDa) and NICD (of 80kDa) (Fig.3c).

Numb-L and Numb-S were [P]-lated at Ser295, in both untreated and GEM Res cells. The amount of Numb-[P]-Ser295 decreased by 30%, in GEM Res cells. The amount of Numb-L and Numb-S-[P]-Ser265 did not change in GEM Res cells compared with untreated cells (Fig.3b). Our results indicate di V erences in Ser295-phosphorylation between GEM Sens and GEM Res cells.

SK-OV-3 cells had more Numb-S than Numb-L. Fur-thermore SK-OV-3 cells had a lower ratio of Numb-L/S-[P]-Ser295 to Numb-L/S than MCF7 cells and ratios of Numb-L/S-[P]-Ser265 to Numb-L/S similar to those in MCF7 cells (Fig.3c).Soluble DLL4 expanded more CD24hi cells from GEM Res cells

The mRNA pro W le of Notch family members and of Notch ligands in MCF7 cells, untreated and treated with chemo-therapeutics, has been reported. MCF7 has lower levels of Notch-ligands of the Delta-like family and higher levels of Notch-ligands of the Jagged-family [39]. MCF7 cells have similar levels of Notch-1 and Notch-2 with normal cells but signi W cantly lower levels of Notch-3 and 4. We investi-gated the e V ects of Notch activation by its ligand, DLL4, on expansion of MCF7 cells. DLL4 expanded more CD24hi than CD24lo cells. DLL4 did not expand CD24hi cells in the presence of GEM (Supplementary Table2). Many rhDLL4-expanded cells were CD44lo CD24lo and CD44lo CD24hi (not shown). Therefore, rhDLL4 did not preferen-tially stimulate proliferation of CD24neg/lo cells.

Naturally immunogenic Notch and Numb peptides expand Ag-speci W c CD8+ T cells

Our results show that a part of Numb-L/S is marked by [P]-lation. These W ndings suggest that cells with CSC-markers can be eliminated by HLA-A2-Notch-1, and Numb-1-pep-tide: complex-speci W c CTL.

Peptides Notch-1 and Numb-1 expanded Notch-1+-TCR+ CD8+ and Numb-1+-TCR+ CD8+ cells from the PBMC of a HLA-A2+ healthy donor (Fig.4a, b). The Numb-1 peptide was more immunogenic than the Notch-1 peptide because it activated more CD8+ cells at 1 M, whereas Notch-1 did so at a concentration of 5 M. Numb-1 peptide-activated PBMC produced IFN-

when incubated with SK-OV-3.A2 cells

Numb-1 and NICD-1 peptide-activated PBMC produced similar amounts of IFN- , in the W rst 24h of co-culture with SK-OV-3.A2 cells. The same levels of IFN- were produced by control peptide, Notch-1-1947, which is not generated by proteasome. The SK-OV-3.A2 cell line acquires expression of HLA-A2 following transfection with a HLA-A2 expression plasmid. IFN- produced by Numb-1-activated cells doubled at 48h of co-culture. The amount of IFN- produced by Notch-1-activated cells did not increase and remained similar to the amount produced by IL-2 activated cells (Fig.4c). Therefore, either SK-OV-3 cells presented more Numb-1 peptide than Notch-1 peptide to CD8+ cells, or Numb-1-CD8+ cells have higher func-tional avidity for HLA-A2-Numb-1 peptide complexes.

To identify whether Numb-degradation is activated by [P]-lation, we repeated the experiment with inhibitors of Ser–Thr-kinases Wortmanin did not inhibit presentation of the Numb-1 peptide, while SB-20380 had a marginal late

e V ect (Fig.4d). The strongest inhibition o

f Numb-1 peptide presentation was mediated by staurosporine, a broad-spec-trum inhibitor of protein–serine–threonine kinase family,indicatin

g that an identi W ed kinase is involved in Numb [P]-lation and degradation.

GEM Res MCF7 cells express more NKG2D ligands than GEM Sens MCF7 cells

To determine whether cells with CSC-markers are sensitive to cellular e V ectors, other than Ag-speci W c CD8+ T cells,we quanti W ed expression of MIC-A/-B in GEM Res , PTX Res and 5-FU Res MCF7 cells. The percentage of MIC-A/B +cells increased by 4.5fold (83.9%) in CD44hi CD24lo GEM Res cells and by threefold (57.5%) in CD44hi CD24lo

PTX Res MCF7 cells (Fig.5a). The percentage of MIC-A/B +CD133+ cells increased from 0.22 in GEM Sens to 6.34 in GEM Res MCF7 cells (not shown). The mean X uorescence intensity values show that the density of MIC-A/B recep-tors per cell was similar in Drug Sens and Drug Res MCF7cells. Therefore, more drug-resistant CSC-like cells will be sensitive to NK/NK-T cells than Drug Sens cells. However,the sensitivity of each CSC-like cell to NK/NK-T cells is not expected to increase compared with Drug Sens cells.Allogeneic Notch and Numb peptide-activated PBMC eliminated cells with CSC-phenotype markers

We investigated whether IL-2-activated, Notch-activated,and Numb-activated allogenic PBMC eliminate cells with

Fig.3a Decreased expression of Notch-1 (NECD ) and Notch-1(TMIC ) in GEM Res MCF7 cells compared with untreated GEM Sens MCF7 cells. One of the two experiments is shown b Numb-L and Numb-S are [P]-lated at Ser 265 and Ser 295 in GEM Sens and GEM Res cells. MCF7 cells contain signi W cantly less Numb-S than SKOV3

cells. c Numb is more [P]-lated at Ser 265 than at Ser 295 NECD was de-tected with mAbs-scc3275 (recognize the whole Notch molecule).H131 mAb detects two TMIC of 100kDa and NICD of 80kDa,respectively. One of two experiments is shown

MCF7

Notch-ER-precursor NECD

Bcl-2

GEM Res

TMIC NICD

β-actin

43.560.4128.956.6

126.879.568.858.9

86.4107.5

188.2179.2

MCF7

SK-OV-3

GEM Sens

GEM Res

GEM Sens

81.521.0

72.021.0

161.0176.7

55.519.2

33.915.2

49.581.7

91.956.993.154.8121.9134.6

Anti-Numb.

Anti-Numb-P-Ser 265

Anti-Numb-P-Ser 295

Notch-1-TMIC Notch-1-NICD

Notch-1/NECD

Notch-1-ER-precursor 120kDa NECD

80 kDa

Numb

P-Numb-S 295P-Numb-S 265

100 kDa Numb-L Numb-S

179.3

OD 151.843.867.7

102.2152.7

150.1123.5

49.8

A B

CSC markers. To account for elimination of cells with CSC markers by allogeneic e V ectors we repeated the experiments, in the presence of IL-2-activated PBMC, and quanti W ed each surviving population of CD44 CD24 cells.Therefore, in addition to allo-recognition of tumor cells by e V ectors, a signi W cant recognition was due to Numb-1 peptide activated T cells . Forty-W ve percent of GEM Res cells had detectable NICD (Fig.5b). Notch-1-positive cells decreased by 68.5% (from 45.4 to 14.3%) after co-culture with Notch-1peptide-activated PBMC (Fig.5d). Numb-1 peptide-activated PBMC decreased the NICD + cells only by 25.3% (from 45.4to 33.9%), whereas IL-2-activated non-speci W c PBMC had no signi W cant e V ect (Fig.5c, e). Therefore, Notch-1-speci W c CD8+ T cells speci W cally eliminated NICD + cells.

To eliminate cells with CSC markers we repeated the experiments and quanti W ed each surviving population of CD44 CD24 cells. To increase stringency of elimination,we used as target MCF7 cells because they expressed less Numb-L and ten times less Numb-S than SK-OV-3 cells.Numb-1-activated T cells were more e V ective than Notch-1 and IL-2-activated e V ectors. Only 10% of the ini-tially plated GEM Res cells survived. The majority of surviv-ing cells were CD44hi CD24lo . Numb-1-activated cells eliminated 2–3 times more CD44hi CD24lo MCF7 cells than other e V ectors (Fig.5f. Columns All and CD44hi CD24lo ).Per e V ecter-cell number, Numb-1-peptide activated cells were more e V ective than IL-2-activated cells, in eliminating CD44hi CD24hi cells (Fig.5f, Column CD44hi CD24hi ).

Discussion

We identi W ed populations in breast and ovarian tumor cell lines which express the phenotypes of breast (ESA +/CD44+/CD24?/lo ) and brain, colon and prostate (CD133+)CSC. Because their function was not yet characterized, we designated these populations as CSC-like. CSC-like cells increased in Drug Res MCF7 and SK-OV-3 cells. Our novel W ndings are: (1) CD24lo population increased in Drug Res cells regardless of the mechanism of drug action; (2) The brain, colon and prostate CSC marker, CD133, is present on cells which express the breast CSC-markers; (3) Cells with CSC-markers/CSC-like cells in GEM Res MCF7 cells were eliminated by Numb-1 peptide-activated PBMC.

GEM and 5-FU are inhibitors of DNA and RNA synthe-sis, which are incorporated in newly synthesized strands.Neither GEM nor 5-FU a V ects cells in G 1 phase [36]. These

R3R4N u m b -1-T C R

6.1

0.70.9B

R3R4

2.3

0.40.3Fig.4Peptides Notch-1 and Numb-1 expanded antigen-speci W c CD8+ cells. a Notch-1-TCR + CD8+ cells expanded by incubation with 5 g Notch-1 (2112–212)and b Numb-1-TCR + CD8+ cells expanded by incubation with 1 g Numb-1 (87–95). TCR hi , TCR med , and TCR lo ,populations are shown in gates R2, R3 and R4. Numbers in each box indicate the percentage of Ag-speci W c cells in the entire population. In the absence of Ag, IL-2 induced expansion of Ag-speci W c cells, was:Notch-1-TCR + cells =(TCR hi : 0.1%, TCR med : 0.1%, TCR lo : 1.0%).Numb-1-TCR + cells =(TCR hi : 0.3%, TCR med : 1.0%, and TCR lo :0.4%) in PBMC from the same donor cultured with IL-2. c SK-OV-3.A2 cells present Numb-1peptide to Numb-1 peptide-activated PB-MC. d Presentation of Numb-1 peptide to Numb-1 peptide-activated cells is dependent on [P]-lation by protein-Ser/Thr-kinases PI3K does not appear to be involved in peptide presentation as shown by lack of e V ect of wortmannin. The MAPK-kinase inhibitor SB20380 had a weak inhibitory e V ect. IFN- was quanti W ed at 24h (Closed symbols )and at 48h (Open symbols ). Numb-1 peptide-activated PBMC pro-duced more IFN- than Notch peptide-activated PBMC. At 48h the amount of IFN- produced by two Notch peptide-activated cell lines was similar with the amount produced by the IL-2-activated cell lines.Only Notch-1 peptide can be presented by HLA-A2 antigens after Notch digestion by proteasome according to the program paproc.de

cells survive because their nucleic acid synthesis is mini-mal. In contrast, PTX acts by epigenetic mechanisms, by interfering with polymerization of actin.

We found only small di V erences in proliferation of CD24? and CD24+ cells when stimulated with medium, and found di V erences when cells were stimulated with DLL4. Cell-cycle analysis indicates that most cells were in G1 phase and only a small fraction less than 5% were in G2M phase (Supplemental Figure1). We used stringent conditions for isolation of these cells by maintaining the drug in culture for several weeks. All non-adherent cells died in these conditions. In clinical practice, the drug given to patient starts decaying and only a small amount is pres-ent several days later. When MCF7 cells were cultured in

Fig.5a The number of MIC-A/ -B+ cells increased in drug-resis-tant MCF7. White peaks repre-sent ESA+ cells. Black peaks represent the MIC-A/B+ CD44+

CD24lo cells. b–e Co-culture of GEM Res MCF7 cells with Notch-1 peptide-activated PBMC decrease the NICD-Notch+ cell numbers. Surviving NICD+ MCF7 cells after co-cul-ture with: b no e V ectors. c IL-2 activated PMBC. d Notch-1 pep-tide-activated PBMC. e Numb-1 peptide-activated PMBC. The % of NICD+ cells is shown in the upper right quadrant. The de-crease in NICD+ cells in relation to the NICD+ cells in panel (b) was: IL-2 activated PBMC, 4.4%; Notch-1-activated PBMC, 68.5%; and Numb-1-activated PBMC, 25.3%. Note: the amount of free NICD in GEM Res MCF7 cells was lower than in UT-MCF7 cells (Fig.3).

f Numb-1 peptide-activated PBMC eliminate CD44+ cells from UT-MCF7 cells. 50,000 GEM Res MCF7 cells were cul-tured with indicated e V ectors for 5days. Then live cells were col-lected and analyzed for expres-sion of CD44 and CD24 in the same experiment. Analysis was performed in large tumor cells of similar cellularity (FS: 800–1000, side scatter 100–800). All indicate all live cells regardless of phenotype, CD44hi CD24lo indicate CSC-like cells, CD44hi CD24hi indicate potentially met-astatic MCF7 cells, CD44lo

CD24lo indicate non-invasive MCF7 cells. Open columns no e V ectors, NP, dotted columns MCF7 cultured with IL-2- activated PBMC, NO, dashed columns left to right MCF7 cultured with Notch-1-activated PBMC, NU, textured columns MCF7 cultured with

Numb-1- activated PBMC

M1M1

MIC A/B

62.487.077.3

18.682.957.554.9

MCF7

UT GEM Res PTX Res5-FU Res

43.4%

33.9%

(

)

Forward scatter

14.3%

45.4%

(

)

All CD44hi

CD24lo

CD44hi

CD24hi

CD44lo

CD24lo

No treatment

No peptide

Numb

Notch

B C

D E

these conditions we counted that only 5–7 clones out of 50,000 drug-treated cells survived for one month and proliferated.

Therefore, we could not establish whether the cells which we analyzed are bona W de CSC, IP or drug-resistant tumor cells, which shared the phenotype of CSC.

Drug Res cells activated autonomous proliferation after drugs were removed. The Notch ligand, DLL4, expanded better CD24hi cells than CD24lo cells. Notch-ligands on neighboring cells activate Notch in Trans ; by endocytosing NECD from responder cells. Free and plastic bound Notch-ligands are weak activators of Notch. Free Notch ligands,can stimulate or inhibit cells in Cis [23]. DLL4 delayed proliferation of CD44lo cells suggesting that it was inhibi-tory as described in other systems [14, 21].

We found that the balance between Notch and its antago-nist, Numb, in Drug Res cancer cells, shifted in favor of Numb. The amount of NECD and TMIC-NICD decreased signi W cantly. The amount of Numb did not change. Numb was present in four forms: non-phosporylated, [P]-lated at Ser 265, [P]-lated at Ser 295 and probably phosphorylated at both positions. Numb-[P]-Ser 295 decreased. Since Numb-1presentation required [P]-lation it is likely that the Numb-1peptide derived from Numb-[P]-Ser 295.

Numb interacts with the aPKC binding partner, PAR-3.Phosphorylation of Numb may inhibit cell migration by inhibiting integrin endocytosis [35]. [P]-Numb does not bind integrins when [P]-lated by atypical protein kinase-C (aPKC). Numb-[P]-Ser 295 recruits 14-3-3 proteins, which inhibit the binding with AP-2 complex in vitro [22].Although the 14-3-3- vector rescued Numb-S from degra-dation in normal MCF-10A cells, Numb-S was not pro-tected by 14-3-3- from degradation in MCF7 cells (Fig.6).In normal cells, the Polo-kinase-1, Plk-1 regulates Numb asymmetry by [P]-lation of Pon [38]. The use of Polo- and Aurora-kinase inhibitors, in cancer treatment, raises the question whether more CSC will remain temporarily quies-cent, or they will activate Notch when Numb decays and accelerate cancer progression. If this is the case, Numb-1

CTL can eliminate Plk-1-inhibitor-surviving cells. When this paper was prepared for publication two independent studies estimated the number of cells with CSC-characteris-tics in MCF7 to 2.4%. MCF7 cells formed mammospheres and required IL-6 to upregulate Notch-3 [7, 29].

In conclusion, we found that Numb-1 speci W c CTL can eliminate CD44hi CD24neg/lo cells. Based on our W ndings,adoptive and active immunotherapy (vaccines) with Notch and Numb should be e V ective in eliminating “quiescent CSC” in patients with breast and ovarian cancer.

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Breast cancer cells expressing stem cell markers

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