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Low Cells
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Oncolytic Adenoviruses Kill Breast Cancer Initiating CD44+CD24–/Low Cells Minna Eriksson1,2, Kilian Guse1,2, Gerd Bauerschmitz1,2,3, Pekka Virkkunen4, Maija Tarkkanen2, Minna Tanner5, Tanja Hakkarainen1,2, Anna Kanerva1,2,6, Renee A Desmond7, Sari Pesonen1,2 and Akseli Hemminki1,2 Cancer Gene Therapy Group, Molecular Cancer Biology Program and Transplantation Laboratory, University of Helsinki, Helsinki, Finland; Department of Oncology and HUSLAB, Helsinki University Central Hospital, Helsinki, Finland; 3Department of Obstetrics and Gynecology, Duesseldorf University Medical Center, Heinrich-Heine University, Duesseldorf, Germany; 4Helsinki Medical Imaging Center, Helsinki University Central Hospital, Helsinki, Finland; 5Institute of Medical Technology, Tampere University and Tampere University Hospital and Department of Oncology, Tampere University Hospital, Tampere, Finland; 6Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland; 7 Comprehensive Cancer Center, Biostatistics and Bioinformatics Unit, University of Alabama at Birmingham, Alabama, USA 1 2
Cancer stem cells have been indicated in the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. In breast cancer, they may reside in the CD44+CD24−/low population. The use of oncolytic adenoviruses presents an attractive anti-tumor approach for eradication of these cells because their entry occurs through infection and they are, therefore, not susceptible to those mechanisms that commonly render stem cells resistant to many drugs. We isolated CD44+CD24−/low cells from patient pleural effusions and confirmed stem cell-like features including oct4 and sox2 expression and Hoechst 33342 exclusion. CD44+CD24−/low cells, including the Hoechst excluding subpopulation, could be effectively killed by oncolytic adenoviruses Ad5/3-∆24 and Ad5.pk7-∆24. In mice, CD44+CD24−/low cells formed orthotopic breast tumors but virus infection prevented tumor formation. Ad5/3∆24 and Ad5.pk7-∆24 were effective against advanced orthotopic CD44+CD24−/low–derived tumors. In summary, Ad5/3-∆24 and Ad5.pk7-∆24 can kill CD44+CD24−/low, and also committed breast cancer cells, making them promising agents for treatment of breast cancer. Received 25 June 2007; accepted 4 August 2007; published online 11 September 2007; doi:10.1038/sj.mt.6300300
Introduction
The CD44+CD24−/low cell population found in many breast cancers exhibits stem cell characteristics, such as self-renewal and differentiation along various mammary epithelial lineages.1–3 In comparison to unsorted cells, a low number of CD44+CD24−/low
cells is sufficient for initiation of tumors in mice.2,4 Further, because of slow replication and capacity for expelling anti-tumor drugs, these cells are resistant to many conventional cancer therapies.5–8 Cancer stem cells may have an important role in cancer relapse following treatment and might therefore be causative of the incurable nature of metastatic breast cancer. Because most modern anti-tumor agents have been approved based on tumor
response, i.e., reduction in the number of differentiated tumor cells which form the bulk of most tumors, agents preferentially active on cancer stem cells may have been missed.9 Oncolytic adenoviruses enter cells through infection and can kill both proliferating and quiescent cells. They are rendered replication deficient in normal cells by engineered genetic changes transcomplemented in tumor cells.10,11 Viral replication leads to oncolytic death of the cell, with the release of thousands of virions that mediate effective intratumoral penetration, and dissemination to distant tumors. One approach for tumor selectivity utilizes a 24 base pair deletion in the retinoblastoma-binding site of adenoviral E1A, which abrogates replication in Rb/p16 intact normal cells as shown previously.10–17 As most, if not all, human cancers are deficient in the Rb/p16 pathway,18 we hypothesized that these viruses would be able to replicate also in cancer stem cells. Because many stem cell types express low levels of the coxsackieadenovirus receptor,19 we utilized capsid modified oncolytic adenoviruses. Ad5/3-∆24 utilizes the serotype 3 receptor highly expressed on many tumor types, Ad5-∆24RGD binds to αvβ integrins, and Ad5.pk7-∆24 can utilize heparan sulfate proteoglycans for entry.10,20–23 Our results suggest that capsid-modified oncolytic adenoviruses may be able to act against cells that are believed to initiate tumors.
Results Previous work suggests that pleural effusions from breast cancer patients might be a convenient source for cancer stem cells.1 Seven of thirteen samples that we obtained contained a CD44+CD24−/low subpopulation, which typically comprised 20–50% of the sample (Figure 1, Supplementary Figure S1). We also found that analysis of primary pleural effusion samples is more difficult than usual, since CD44+CD24−/low cells do not attach and could not be passaged in vitro. A feature suggested for both tumor and normal tissue stem cells is a membrane transporter–mediated capacity for Hoechst 33342 exclusion, which might serve as a useful surrogate marker for resistance to lipophilic anti-cancer agents.24,25 CD44+CD24−/low sorting enriched this subpopulation from 1% (±1.1%) to 7%
Correspondence: Akseli Hemminki, University of Helsinki, Haartmaninkatu 8, Biomedicum, 00014 Helsinki, Finland. E-mail: [email protected]
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Figure 1 Isolation of putative breast cancer stem cells. CD44+CD24−/low cells were collected from pleural effusions of breast cancer patients using magnetic beads. The expression of CD44 and CD24 in (a) unsorted, (b) CD44−CD24−/low, and (c) CD44+D24−/low cells was analyzed by flow cytometry.
(±2.1%) (N = 3, P = 0.0001, Figure 2a and b). It is tantalizing to speculate that the cell population capable of dye exclusion might harbor a higher proportion of actual stem cells as opposed to progenitors, which probably also reside in the CD44+CD24−/low
population. To further evaluate the possible stem cell characteristics of the CD44+CD24−/low population, expression of oct4 and sox2 was studied by means of a semi-quantitative reverse transcriptase polymerase chain reaction analysis (real-time-PCR).26,27 We were surprised to find an unequal amplification of β-actin, which had been included as a control for the amount of total RNA. Total RNA was carefully measured before the real-time-PCR analysis was done and equal amounts of template were used. The PCR was done several times in different conditions and the same result was obtained every time. This suggests that β-actin may not be the optimal control to use when analyzing putative stem cells. A literature search indicated that others have had the same experience.28,29 Nevertheless, the main question we tried to answer by means of the real-time-PCR analysis was a qualitative one relating to oct4 and sox2 expression; therefore, the non-reliability of β-actin did not interfere with our interpretation of the results. All samples were positive for oct4 and sox2, suggesting that when a CD44+CD24−/low population is present, it contains stem cell-like cells (Figure 2c). Oct4 and CD44 protein expression was confirmed by immunofluorescence staining (Figure 2d). When unsorted pleural effusion cells were infected, Ad5/3∆24 killed most cells rapidly, and Ad5.pK7-∆24 killed all cells in the sample (Figure 3a). Next, we focused on the CD44+CD24−/low population. Ad5/3-∆24 and Ad5.pK7-∆24 killed all cells in most samples (Figure 3b–d, Supplementary Figure S2). Optimal Molecular Therapy vol. 15 no. 12 dec. 2007
Figure 2 Stem cell features of pleural effusion cells. (a) Unsorted and (b) CD44+CD24−/low cells were stained with Hoechst 33342 and imaged by fluorescence microscope. A subpopulation of CD44+D24−/low cells exclude Hoechst dye (arrows). (c) The expression of stem cell markers oct4 and sox2 in CD44+CD24−/low–sorted cells was confirmed by semiquantitative real-time polymerase chain reaction. Fat tissue–derived mesenchymal stem cells were used as a positive control (+) and β-actin as a control for total RNA. The pleural effusion samples are indicated with sample number. (d) Immunofluorescence staining of Oct-4 (red) and CD44 (green) expression in the CD44+CD24−/low cell population.
timing of the viability assay with these cells was not easy because cytopathic effects cannot be seen when cells do not adhere. It is possible that with more time, Ad5/3-∆24 and Ad5.pK7-∆24 might have been able to kill all the cells in all the samples. Although Ad5-∆24RGD showed activity in some samples, Ad5/3-∆24 and Ad5.pK7-∆24 emerged as the most promising agents. Ad300wt, a serotype 5 adenovirus, was included as a wild-type control. However, in vitro data quickly suggested that capsid modification could be useful for optimal oncolytic potency. To confirm that capsid-modified oncolytic viruses can completely eradicate the CD44+CD24−/low population, trypan blue staining was performed (Figure 3e). Approximately 85% of the uninfected cells were alive after 8 days, whereas none of the cells infected with Ad5/3-∆24 and Ad5.pk7-∆24 were alive. Initially, a pleural effusion sample sorted for CD44+CD24−/low was injected into eight mammary fat pads of nude mice but no tumors developed in a follow-up of 7 months. We then started to use non-obese diabetic/severe combined immunodeficient mice and also treated mice with human estrogen. Our hypotheses were that the absence of B-cells might facilitate tumor growth and also that putative breast cancer stem cells might have arisen from hormonically active breast epithelium; therefore, estrogen might increase either the growth or initiation rate of CD44+/CD24− derived tumors. All non-obese diabetic/severe combined immunodeficient mice injected with unsorted or CD44+CD24−/low cells from a breast cancer patient developed tumors by day 95. The first tumor in mice injected with CD24+ cells appeared on day 113 (Figure 4), confirming previous data that sorting for CD44+CD24−/low also selects for a population capable of forming tumors rapidly. When cells were infected with Ad5/3-∆24, no tumors grew (Figure 4). 2089
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Figure 3 Killing of CD44+CD24−/low cells with oncolytic adenoviruses. (a–d) Cell killing was measured with MTS assay (Promega, Madison, WI). Positive and negative controls were wild-type adenovirus Ad300wt and replication deficient Ad5luc1. VP = viral particles. The value for non-infected cells was set as 100%. Data are expressed as means ± SEM. (e) The absence of living cells following infection with oncolytic viruses Ad5/3-∆24 and Ad5.pK7-∆24 was confirmed by trypan blue exclusion 8 days after infection.
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Figure 4 Anti-tumor efficacy of Ad5/3-∆24 against CD44+CD24low/−–derived tumors. (a) Injection of CD44+CD24low/− cells fresh from patients resulted in tumors in all animals (N = 10 tumors in five mice), while pre-infection (filled symbols, N = 4 tumors in two mice), prevented growth in all cases. Size of tumors detected only after skin removal was set as 50 mm3. (b) CD44+CD24low/− JIMT-1 cells formed tumors in all mammary fat pads (N = 6 tumors in three mice) while pre-infection (filled symbols, N = 4 tumors in two mice) prevented tumor growth. Treatment of established tumors with 3 × 109 viral particles of Ad5/3∆24 (arrows) stopped tumor growth and prevented death of mice.
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Figure 5 Anti-tumor efficacy of oncolytic viruses against CD44+CD24low/−– derived tumors. Injection of 2 × 106 cells into mammary fat pads of nonobese diabetic/severe combined immunodeficient mice resulted in tumors in 14 days (N = 8 tumors in four mice/group, i.e., total 40 tumors in 20 mice). Viruses or just medium were then injected thrice weekly (arrows) and tumor size was calculated: length × width2 × 0.5. All viruses gave an anti-tumor effect when compared to medium only (all P < 0.0001).
Intriguingly, all mice injected with uninfected cells in both the upper mammary fat pads developed a large tumor on one side and a small one on the other. Injection of CD44+/CD24−/low JIMT-1 cells (a low passage pleural effusion explant) into the mammary fat pads of nonobese diabetic/severe combined immunodeficient mice resulted in tumor growth, but when cells were treated with Ad5/3-∆24, no tumors formed (Figure 4). To analyze the effect of Ad5/3∆24 on orthotopic pre-terminal CD44+/CD24−/low–derived disease, tumors were allowed to grow as large as animal regulations allowed. Then, intratumoral virus injections were performed, and abrogation of tumor growth was seen (Figure 4b). Without treatment, carrying out killing of these mice would have been required on day 35, but now they remained alive until the end of the experiment (day 58). In a larger subsequent experiment, Ad5/3-∆24 and Ad5.pK7-∆24 gave significant anti-tumor efficacy versus mock (both P < 0.0001, Figure 5). In the culture, 10% of JIMT-1 cells were CD44+CD24−/low. After sorting, 100% of cells injected into mice were CD44+CD24−/low, but when tumors were removed on day 48, the CD44+CD24−/low cell proportion had returned to 11%. After virus injections, 6.4% (Ad300wt), 5.0% (Ad5/3-d24), 1.1% (Ad5.pK7-d24), and 3.8% (Ad5-d24RGD) of cells were of CD44+CD24−/low type. 14% (±3.4%) versus 3.0% (±0.9%) of cells in the CD44+CD24−/low population of untreated versus treated tumors were capable of Hoechst exclusion (Mann–Whitney P < 0.001).
Discussion Cancer stem cells have been suspected to underlie the incurable and relapsing nature of many metastatic tumors. Although cell populations suspected to contain stem cells have been mainly indicated, there is no unequivocal consensus on how to conclusively identify the most relevant cells. There are a number of hypotheses on how tumor-initiating cells might actually arise; this includes the possibility of mutation of normal tissue stem or progenitor cells, “mutator” phenotypes, and reversible epithelial–mesenchymal transition. With regard to breast cancer, previous work has nevertheless suggested that tumor-initiating Molecular Therapy vol. 15 no. 12 dec. 2007
cells may reside in the CD44+CD24−/low population.1–3 In this study we have confirmed that CD44+CD24−/low cells can be purified from breast cancer–related pleural fluid, and that stem cell features are present in those cells. An intriguing subpopulation is capable of excluding Hoechst dye, which may be a surrogate for resistance to many anti-cancer drugs. Sorting for CD44+CD24−/low increased the proportion of Hoechst excluding cells sevenfold. After three rounds of enrichment [(i) accumulation in pleural effusion, (ii) sorting for CD44+, (iii) sorting for CD24−/low], ~7% of the remaining cells had the capacity for Hoechst dye exclusion. Further studies are needed to investigate if true stem cells reside in this population. Capsid-modified oncolytic viruses Ad5.pK7-∆24 and Ad5/3-∆24 seemed effective in the killing of CD44+CD24−/low cells in vitro and in vivo, including the Hoechst excluding subpopulation. When established CD44+CD24−/low–derived tumors were treated, tumor growth could be abrogated, but complete tumor eradication was not seen in most mice. This suggests that CD44+CD24−/low–derived tumors are relatively treatment resistant. Nevertheless, the same is true for many cell line–derived tumors and suggested reasons include intratumoral complexities such as stromal areas, hyperbaric, hypoxic, and necrotic areas.11,15,19,30–37 As an alternative hypothesis, the lack of complete eradication of CD44+CD24−/low–derived tumors might be explained in part by killing of only differentiated tumor cells while CD44+CD24−/low would remain resistant and their proportion would decrease because of differentiation into sensitive tumor cells. However, the lack of tumor formation following an injection of infected CD44+CD24−/low cells (Figure 4), taken together with the in vitro oncolysis data (Figure 3), seems to suggest a possible replication in and lysis of CD44+CD24−/low cells by oncolytic adenovirus in vivo as well. When CD44+CD24−/low cells were injected into mice, tumors formed rapidly. Interestingly, even though 100% CD44+CD24−/low cells were injected, the proportion of these cells returned to 11% after a tumor had developed (in untreated mice). This supports the notion of asymmetric cell division, a feature associated with stem cells. Further, treatment with Ad5.pK7-∆24 and Ad5/3-∆24 reduced the proportion of CD44+CD24−/low and Hoechst excluding cells in those tumors, suggesting activity against cells suspected as initiating tumors, metastases or relapse. In conclusion, much further work is required to understand and analyze these elusive cells, which may be important with regard to curing advanced cancers. Ultimately, clinical trials will be needed to study if Ad5.pK7-∆24 and Ad5/3-∆24 can enhance the survival of breast cancer patients. However, promising preclinical data has been obtained using both viruses in breast cancer cell lines, clinical specimens, and orthotopic animal models of both locally advanced and metastatic breast cancer.22,23 Therefore, these viruses may be effective in the killing of both “differentiated” and “tumor initiating” breast cancer cells, which might in turn be useful for improving patient survival. Further, the favorable (preliminary) biodistribution, and toxicity data set the stage for clinical translation.23 Because in vitro work and animal models cannot fully recreate the human environment, clinical studies will be needed to ultimately demonstrate the safety of these agents with regard to normal human stem cells and other normal tissues. 2091
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Finally, improved understanding of cancer stem cells, if indeed they exist, may have tremendous relevance for how cancer should be treated. It is ominous that cancer stem cells have been reported resistant to almost all anti-tumor approaches that have already been established for the treatment of metastatic disease such as ionizing radiation, hormonal therapy, chemotherapy, and small molecular inhibitors.5,7,38,39 Nevertheless, it is promising that our results suggest that they may be susceptible to capsid-modified oncolytic adenoviruses.
tumors in four mice in each group, i.e., totally 40 tumors in 20 mice. The quantity of CD44+CD24−/low cells available limited the number of mice that could be included in the experiments. Mice were also injected with 1 mg/kg Estradurin (Pfizer, New York, NY) every 3 weeks throughout the experiment. Tumors were measured with a caliper two to three times/ week. Intratumoral injections were performed with 3 × 109 viral particles of indicated viruses at time points shown by arrows in figures. Tumor volume was calculated: length × width2 × 0.5. The Provincial Government of Southern Finland approved the animal experiments.
Materials and methods
9.1; SAS, Cary, NC) was used for comparison of tumor sizes. Data was logtransformed for normality. The effects of the treatment group, time and the interaction between the treatment group time were evaluated by F tests. Curvature in the growth curves was tested by a quadratic term for time. A priori planned comparisons of differences in the predicted treatment means of all groups to mock were computed by Tukey–Kramer adjusted two-sided t-statistics averaged over all time points.
Isolation of breast cancer stem cells. Pleural effusions were obtained
(with ethics committee approval and after obtaining an informed consent) directly from thoracocentesis and washed with Dulbecco’s modified Eagle’s medium-F12 supplemented with 10 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 µg/ml insulin, and 0.4% bovine serum albumin (all from Sigma, St. Louis, MO). JIMT-1 cells were cultured according to normal conditions. Cells were sorted with fluorescein isothiocyanate–labeled anti-CD44 and phycoerythrin-labeled anti-CD24 antibodies (BD Pharmingen, Franklin Lakes, NJ), which were collected with fluorescein isothiocyanate- and phycoerythrin-conjugated magnetic beads, respectively (Miltenyi Biotech, Bergisch Gladbach, Germany). The sorted cell populations were confirmed to be CD24 negative and CD44 positive by flow cytometry. Both unsorted and CD44+CD24−/low living cell populations were stained with Hoechst 33342 (5 µg/ml; Sigma, St. Louis, MO) at 37 °C, mounted on glass slides and viewed under a fluorescence microscope. Expression of stem cell markers. Messenger RNA was isolated using
RNAeasyTM (Qiagen, Hilden, Germany). The following primers were used for real-time-PCR; oct4 forward 5′-CGCACCACTGGCATTG TCAT-3′, reverse 5′-TTCTCCTTGATGTCACGCAC-3′, sox2 forward 5′-GGCAGCTACGCATGATGCAGGAGC-3′, reverse 5′-CTGGTCATG GAGTTGTACTGCACG-3′ β-actin forward 5′-CGAGGCCCAGAGCA AGACA-3′, reverse 5′-CACAGCTTCTCCTTAATGTCACG-3′. Immu nofluorescence staining of paraformaldehyde-fixed CD44+CD24−/low cells was performed with Oct3/4 and CD44 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; BD Pharmingen, Franklin Lakes, NJ). Oncolytic adenoviruses. Viruses used have been described in detail previ-
ously: Ad5-∆24E3+ (ref. 10), Ad5/3-∆24 (ref. 20), Ad5-∆24RGD (ref. 21), Ad5.pk7-∆24 (ref. 23). Positive and negative controls were wild-type adenovirus Ad300wt (ATCC VR-5) and replication deficient Ad5luc1 (ref. 21). Viruses were produced, purified, validated, and quality controlled according to standard methods.11 Cytotoxicity assays. Cell viability was measured 5–14 days after tripli-
cate infection utilizing CellTiter 96 AQueous One Solution cell proliferation Assay (Promega, Madison, WI), which measures mitochondrial activity as a surrogate for the number of live cells. Assays were performed 8 days (PL1, PL4, and JIMT-1), 14 days (PL8), and 12 days (PL11 and PL13) after virus infection. Samples used in cytotoxicity assays contained 100% CD44+CD24−/low cells because virus infection was performed after sorting, except PL1, which was analyzed without sorting. The complete PL1 sample was used for infection and therefore the proportion of CD44+CD24−/low cell could not be analyzed. The total number and proportion of viable cells in patient sample PL11 was also studied with trypan blue staining (Invitrogen, Carlsbad, CA) 8 days after infection. In vivo studies. 2 × 106 sorted or unsorted patient pleural effusion or JIMT-1
cells were injected together with Matrigel (BD Pharmingen, Franklin Lakes, NJ) into the uppermost mammary fat pads of non-obese diabetic/severe combined immunodeficient mice. Figure 4a: N = 14 tumors in seven mice. Figure 4b: N = 10 tumors in five mice. Figure 5: N = 8
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Statistics. The repeated measures growth model in PROC MIXED (SAS
Acknowledgments We thank Eerika Karli (University of Helsinki and Helsinki University Central Hospital) and Aila Karioja-Kallio (University of Helsinki and Helsinki University Central Hospital) for their superb technical assistance. This study was supported by the European Union framework project 6 THERADPOX and APOTHERAPY, Helsinki University Central Hospital Research Funds, Sigrid Juselius Foundation, Academy of Finland, Emil Aaltonen Foundation, Finnish Cancer Society, University of Helsinki, the Finnish Oncology Association, Helsinki Graduate School in Biotechnology and Molecular Biology and the Finnish Cultural Foundation. A.H. is K. Albin Johansson Research Professor of the Foundation for the Finnish Cancer Institute. No conflicts of interest apply.
Supplementary Material Figure S1. Additional samples analyzed with FACS as described in legend for Figure 1. Figure S2. Oncolysis of CD44+CD24−/low cells from additional samples was analyzed as in Figure 3.
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13. Howe, JA, Demers, GW, Johnson, DE, Neugebauer, SE, Perry, ST, Vaillancourt, MT et al. (2000). Evaluation of E1-mutant adenoviruses as conditionally replicating agents for cancer therapy. Mol Ther 2: 485–495. 14. Sauthoff, H, Pipiya, T, Heitner, S, Chen, S, Bleck, B, Reibman, J et al. (2004). Impact of E1a modifications on tumor-selective adenoviral replication and toxicity. Mol Ther 10: 749–757. 15. Kangasniemi, L, Kiviluoto, T, Kanerva, A, Raki, M, Ranki, T, Sarkioja, M et al. (2006). Infectivity-enhanced adenoviruses deliver efficacy in clinical samples and orthotopic models of disseminated gastric cancer. Clin Cancer Res 12: 3137–3144. 16. Stoff-Khalili, MA, Rivera, AA, Le, LP, Stoff, A, Everts, M, Contreras, JL et al. (2006). Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents. Cancer Gene Ther 13: 606–618. 17. Kanerva, A, Raki, M, Ranki, T, Sarkioja, M, Koponen, J, Desmond, RA et al. (2007). Chlorpromazine and apigenin reduce adenovirus replication and decrease replication associated toxicity. J Gene Med 9: 3–9. 18. Sherr, CJ (1996). Cancer cell cycles. Science 274: 1672–1677. 19. Hakkarainen, T, Sarkioja, M, Lehenkari, P, Miettinen, S, Ylikomi, T, Suuronen, R et al. (2007). Human mesenchymal stem cells lack tumor tropism but enhance the antitumor activity of oncolytic adenoviruses in orthotopic lung and breast tumors. Hum Gene Ther 18: 627–641. 20. Kanerva, A, Zinn, KR, Chaudhuri, TR, Lam, JT, Suzuki, K, Uil, TG et al. (2003). Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus. Mol Ther 8: 449–458. 21. Suzuki, K, Fueyo, J, Krasnykh, V, Reynolds, PN, Curiel, DT and Alemany, R (2001). A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency. Clin Cancer Res 7: 120–126. 22. Ranki, T, Sarkioja, M, Hakkarainen, T, von Smitten, K, Kanerva, A and Hemminki, A (2007). Systemic efficacy of oncolytic adenoviruses in imagable orthotopic models of hormone refractory metastatic breast cancer. Int J Cancer 121: 165–174. 23. Ranki, T, Kanerva, A, Ristimaki, A, Hakkarainen, T, Sarkioja, M, Kangasniemi, L et al. (2007). A heparan sulfate-targeted conditionally replicative adenovirus, Ad5.pk7-Delta24, for the treatment of advanced breast cancer. Gene Ther 14: 58–67. 24. Zhou, S, Schuetz, JD, Bunting, KD, Colapietro, AM, Sampath, J, Morris, JJ et al. (2001). The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7: 1028–1034. 25. Dean, M, Fojo, T and Bates, S (2005). Tumour stem cells and drug resistance. Nat Rev Cancer 5: 275–284. 26. Nichols, J, Zevnik, B, Anastassiadis, K, Niwa, H, Klewe-Nebenius, D, Chambers, I et al. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95: 379–391.
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27. Zhao, S, Nichols, J, Smith, AG and Li, M (2004). SoxB transcription factors specify neuroectodermal lineage choice in ES cells. Mol Cell Neurosci 27: 332–342. 28. Lee, BC, Cheng, T, Adams, GB, Attar, EC, Miura, N, Lee, SB et al. (2003). P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells. Genes Dev 17: 1592–1604. 29. Cowan, KH, Moscow, JA, Huang, H, Zujewski, JA, O’Shaughnessy, J, Sorrentino, B et al. (1999). Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients. Clin Cancer Res 5: 1619–1628. 30. Shayakhmetov, DM, Li, ZY, Ni, S and Lieber, A (2002). Targeting of adenovirus vectors to tumor cells does not enable efficient transduction of breast cancer metastases. Cancer Res 62: 1063–1068. 31. Hemminki, A, Zinn, KR, Liu, B, Chaudhuri, TR, Desmond, RA, Rogers, BE et al. (2002). In vivo molecular chemotherapy and noninvasive imaging with an infectivity-enhanced adenovirus. J Natl Cancer Inst 94: 741–749. 32. Sauthoff, H, Hu, J, Maca, C, Goldman, M, Heitner, S, Yee, H et al. (2003). Intratumoral spread of wild-type adenovirus is limited after local injection of human xenograft tumors: virus persists and spreads systemically at late time points. Hum Gene Ther 14: 425–433. 33. Raki, M, Rein, DT, Kanerva, A and Hemminki, A (2006). Gene transfer approaches for gynecological diseases. Mol Ther 14: 154–163. 34. Raki, M, Hakkarainen, T, Bauerschmitz, GJ, Sarkioja, M, Desmond, RA, Kanerva, A et al. (2007). Utility of TK/GCV in the context of highly effective oncolysis mediated by a serotype 3 receptor targeted oncolytic adenovirus. Gene Ther (epub ahead of print). 35. Rajecki, M, Kanerva, A, Stenman, UH, Tenhunen, M, Kangasniemi, L, Sarkioja, M et al. (2007). Treatment of prostate cancer with Ad5/3Delta24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo. Mol Cancer Ther 6: 742–751. 36. Guse, K, Dias, JD, Bauerschmitz, GJ, Hakkarainen, T, Aavik, E, Ranki, T et al. (2007). Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo. Gene Ther 14: 902–911. 37. Sarkioja, M, Kanerva, A, Salo, J, Kangasniemi, L, Eriksson, M, Raki, M et al. (2006). Noninvasive imaging for evaluation of the systemic delivery of capsid-modified adenoviruses in an orthotopic model of advanced lung cancer. Cancer 107: 1578–1588. 38. Phillips, TM, McBride, WH and Pajonk, F (2006). The response of CD24−/low/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 98: 1777–1785. 39. Brendel, C, Scharenberg, C, Dohse, M, Robey, RW, Bates, SE, Shukla, S et al. (2007). Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells. Leukemia 21: 1267–1275.
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Oncolytic Adenoviruses Kill Breast Cancer Initiating CD44+CD24–/Low Cells Minna Eriksson1,2, Kilian Guse1,2, Gerd Bauerschmitz1,2,3, Pekka Virkkunen4, Maija Tarkkanen2, Minna Tanner5, Tanja Hakkarainen1,2, Anna Kanerva1,2,6, Renee A Desmond7, Sari Pesonen1,2 and Akseli Hemminki1,2 Cancer Gene Therapy Group, Molecular Cancer Biology Program and Transplantation Laboratory, University of Helsinki, Helsinki, Finland; Department of Oncology and HUSLAB, Helsinki University Central Hospital, Helsinki, Finland; 3Department of Obstetrics and Gynecology, Duesseldorf University Medical Center, Heinrich-Heine University, Duesseldorf, Germany; 4Helsinki Medical Imaging Center, Helsinki University Central Hospital, Helsinki, Finland; 5Institute of Medical Technology, Tampere University and Tampere University Hospital and Department of Oncology, Tampere University Hospital, Tampere, Finland; 6Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland; 7 Comprehensive Cancer Center, Biostatistics and Bioinformatics Unit, University of Alabama at Birmingham, Alabama, USA 1 2
Cancer stem cells have been indicated in the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. In breast cancer, they may reside in the CD44+CD24−/low population. The use of oncolytic adenoviruses presents an attractive anti-tumor approach for eradication of these cells because their entry occurs through infection and they are, therefore, not susceptible to those mechanisms that commonly render stem cells resistant to many drugs. We isolated CD44+CD24−/low cells from patient pleural effusions and confirmed stem cell-like features including oct4 and sox2 expression and Hoechst 33342 exclusion. CD44+CD24−/low cells, including the Hoechst excluding subpopulation, could be effectively killed by oncolytic adenoviruses Ad5/3-∆24 and Ad5.pk7-∆24. In mice, CD44+CD24−/low cells formed orthotopic breast tumors but virus infection prevented tumor formation. Ad5/3∆24 and Ad5.pk7-∆24 were effective against advanced orthotopic CD44+CD24−/low–derived tumors. In summary, Ad5/3-∆24 and Ad5.pk7-∆24 can kill CD44+CD24−/low, and also committed breast cancer cells, making them promising agents for treatment of breast cancer. Received 25 June 2007; accepted 4 August 2007; published online 11 September 2007; doi:10.1038/sj.mt.6300300
Introduction
The CD44+CD24−/low cell population found in many breast cancers exhibits stem cell characteristics, such as self-renewal and differentiation along various mammary epithelial lineages.1–3 In comparison to unsorted cells, a low number of CD44+CD24−/low
cells is sufficient for initiation of tumors in mice.2,4 Further, because of slow replication and capacity for expelling anti-tumor drugs, these cells are resistant to many conventional cancer therapies.5–8 Cancer stem cells may have an important role in cancer relapse following treatment and might therefore be causative of the incurable nature of metastatic breast cancer. Because most modern anti-tumor agents have been approved based on tumor
response, i.e., reduction in the number of differentiated tumor cells which form the bulk of most tumors, agents preferentially active on cancer stem cells may have been missed.9 Oncolytic adenoviruses enter cells through infection and can kill both proliferating and quiescent cells. They are rendered replication deficient in normal cells by engineered genetic changes transcomplemented in tumor cells.10,11 Viral replication leads to oncolytic death of the cell, with the release of thousands of virions that mediate effective intratumoral penetration, and dissemination to distant tumors. One approach for tumor selectivity utilizes a 24 base pair deletion in the retinoblastoma-binding site of adenoviral E1A, which abrogates replication in Rb/p16 intact normal cells as shown previously.10–17 As most, if not all, human cancers are deficient in the Rb/p16 pathway,18 we hypothesized that these viruses would be able to replicate also in cancer stem cells. Because many stem cell types express low levels of the coxsackieadenovirus receptor,19 we utilized capsid modified oncolytic adenoviruses. Ad5/3-∆24 utilizes the serotype 3 receptor highly expressed on many tumor types, Ad5-∆24RGD binds to αvβ integrins, and Ad5.pk7-∆24 can utilize heparan sulfate proteoglycans for entry.10,20–23 Our results suggest that capsid-modified oncolytic adenoviruses may be able to act against cells that are believed to initiate tumors.
Results Previous work suggests that pleural effusions from breast cancer patients might be a convenient source for cancer stem cells.1 Seven of thirteen samples that we obtained contained a CD44+CD24−/low subpopulation, which typically comprised 20–50% of the sample (Figure 1, Supplementary Figure S1). We also found that analysis of primary pleural effusion samples is more difficult than usual, since CD44+CD24−/low cells do not attach and could not be passaged in vitro. A feature suggested for both tumor and normal tissue stem cells is a membrane transporter–mediated capacity for Hoechst 33342 exclusion, which might serve as a useful surrogate marker for resistance to lipophilic anti-cancer agents.24,25 CD44+CD24−/low sorting enriched this subpopulation from 1% (±1.1%) to 7%
Correspondence: Akseli Hemminki, University of Helsinki, Haartmaninkatu 8, Biomedicum, 00014 Helsinki, Finland. E-mail: [email protected]
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(±2.1%) (N = 3, P = 0.0001, Figure 2a and b). It is tantalizing to speculate that the cell population capable of dye exclusion might harbor a higher proportion of actual stem cells as opposed to progenitors, which probably also reside in the CD44+CD24−/low
population. To further evaluate the possible stem cell characteristics of the CD44+CD24−/low population, expression of oct4 and sox2 was studied by means of a semi-quantitative reverse transcriptase polymerase chain reaction analysis (real-time-PCR).26,27 We were surprised to find an unequal amplification of β-actin, which had been included as a control for the amount of total RNA. Total RNA was carefully measured before the real-time-PCR analysis was done and equal amounts of template were used. The PCR was done several times in different conditions and the same result was obtained every time. This suggests that β-actin may not be the optimal control to use when analyzing putative stem cells. A literature search indicated that others have had the same experience.28,29 Nevertheless, the main question we tried to answer by means of the real-time-PCR analysis was a qualitative one relating to oct4 and sox2 expression; therefore, the non-reliability of β-actin did not interfere with our interpretation of the results. All samples were positive for oct4 and sox2, suggesting that when a CD44+CD24−/low population is present, it contains stem cell-like cells (Figure 2c). Oct4 and CD44 protein expression was confirmed by immunofluorescence staining (Figure 2d). When unsorted pleural effusion cells were infected, Ad5/3∆24 killed most cells rapidly, and Ad5.pK7-∆24 killed all cells in the sample (Figure 3a). Next, we focused on the CD44+CD24−/low population. Ad5/3-∆24 and Ad5.pK7-∆24 killed all cells in most samples (Figure 3b–d, Supplementary Figure S2). Optimal Molecular Therapy vol. 15 no. 12 dec. 2007
Figure 2 Stem cell features of pleural effusion cells. (a) Unsorted and (b) CD44+CD24−/low cells were stained with Hoechst 33342 and imaged by fluorescence microscope. A subpopulation of CD44+D24−/low cells exclude Hoechst dye (arrows). (c) The expression of stem cell markers oct4 and sox2 in CD44+CD24−/low–sorted cells was confirmed by semiquantitative real-time polymerase chain reaction. Fat tissue–derived mesenchymal stem cells were used as a positive control (+) and β-actin as a control for total RNA. The pleural effusion samples are indicated with sample number. (d) Immunofluorescence staining of Oct-4 (red) and CD44 (green) expression in the CD44+CD24−/low cell population.
timing of the viability assay with these cells was not easy because cytopathic effects cannot be seen when cells do not adhere. It is possible that with more time, Ad5/3-∆24 and Ad5.pK7-∆24 might have been able to kill all the cells in all the samples. Although Ad5-∆24RGD showed activity in some samples, Ad5/3-∆24 and Ad5.pK7-∆24 emerged as the most promising agents. Ad300wt, a serotype 5 adenovirus, was included as a wild-type control. However, in vitro data quickly suggested that capsid modification could be useful for optimal oncolytic potency. To confirm that capsid-modified oncolytic viruses can completely eradicate the CD44+CD24−/low population, trypan blue staining was performed (Figure 3e). Approximately 85% of the uninfected cells were alive after 8 days, whereas none of the cells infected with Ad5/3-∆24 and Ad5.pk7-∆24 were alive. Initially, a pleural effusion sample sorted for CD44+CD24−/low was injected into eight mammary fat pads of nude mice but no tumors developed in a follow-up of 7 months. We then started to use non-obese diabetic/severe combined immunodeficient mice and also treated mice with human estrogen. Our hypotheses were that the absence of B-cells might facilitate tumor growth and also that putative breast cancer stem cells might have arisen from hormonically active breast epithelium; therefore, estrogen might increase either the growth or initiation rate of CD44+/CD24− derived tumors. All non-obese diabetic/severe combined immunodeficient mice injected with unsorted or CD44+CD24−/low cells from a breast cancer patient developed tumors by day 95. The first tumor in mice injected with CD24+ cells appeared on day 113 (Figure 4), confirming previous data that sorting for CD44+CD24−/low also selects for a population capable of forming tumors rapidly. When cells were infected with Ad5/3-∆24, no tumors grew (Figure 4). 2089
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Figure 4 Anti-tumor efficacy of Ad5/3-∆24 against CD44+CD24low/−–derived tumors. (a) Injection of CD44+CD24low/− cells fresh from patients resulted in tumors in all animals (N = 10 tumors in five mice), while pre-infection (filled symbols, N = 4 tumors in two mice), prevented growth in all cases. Size of tumors detected only after skin removal was set as 50 mm3. (b) CD44+CD24low/− JIMT-1 cells formed tumors in all mammary fat pads (N = 6 tumors in three mice) while pre-infection (filled symbols, N = 4 tumors in two mice) prevented tumor growth. Treatment of established tumors with 3 × 109 viral particles of Ad5/3∆24 (arrows) stopped tumor growth and prevented death of mice.
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Intriguingly, all mice injected with uninfected cells in both the upper mammary fat pads developed a large tumor on one side and a small one on the other. Injection of CD44+/CD24−/low JIMT-1 cells (a low passage pleural effusion explant) into the mammary fat pads of nonobese diabetic/severe combined immunodeficient mice resulted in tumor growth, but when cells were treated with Ad5/3-∆24, no tumors formed (Figure 4). To analyze the effect of Ad5/3∆24 on orthotopic pre-terminal CD44+/CD24−/low–derived disease, tumors were allowed to grow as large as animal regulations allowed. Then, intratumoral virus injections were performed, and abrogation of tumor growth was seen (Figure 4b). Without treatment, carrying out killing of these mice would have been required on day 35, but now they remained alive until the end of the experiment (day 58). In a larger subsequent experiment, Ad5/3-∆24 and Ad5.pK7-∆24 gave significant anti-tumor efficacy versus mock (both P < 0.0001, Figure 5). In the culture, 10% of JIMT-1 cells were CD44+CD24−/low. After sorting, 100% of cells injected into mice were CD44+CD24−/low, but when tumors were removed on day 48, the CD44+CD24−/low cell proportion had returned to 11%. After virus injections, 6.4% (Ad300wt), 5.0% (Ad5/3-d24), 1.1% (Ad5.pK7-d24), and 3.8% (Ad5-d24RGD) of cells were of CD44+CD24−/low type. 14% (±3.4%) versus 3.0% (±0.9%) of cells in the CD44+CD24−/low population of untreated versus treated tumors were capable of Hoechst exclusion (Mann–Whitney P < 0.001).
Discussion Cancer stem cells have been suspected to underlie the incurable and relapsing nature of many metastatic tumors. Although cell populations suspected to contain stem cells have been mainly indicated, there is no unequivocal consensus on how to conclusively identify the most relevant cells. There are a number of hypotheses on how tumor-initiating cells might actually arise; this includes the possibility of mutation of normal tissue stem or progenitor cells, “mutator” phenotypes, and reversible epithelial–mesenchymal transition. With regard to breast cancer, previous work has nevertheless suggested that tumor-initiating Molecular Therapy vol. 15 no. 12 dec. 2007
cells may reside in the CD44+CD24−/low population.1–3 In this study we have confirmed that CD44+CD24−/low cells can be purified from breast cancer–related pleural fluid, and that stem cell features are present in those cells. An intriguing subpopulation is capable of excluding Hoechst dye, which may be a surrogate for resistance to many anti-cancer drugs. Sorting for CD44+CD24−/low increased the proportion of Hoechst excluding cells sevenfold. After three rounds of enrichment [(i) accumulation in pleural effusion, (ii) sorting for CD44+, (iii) sorting for CD24−/low], ~7% of the remaining cells had the capacity for Hoechst dye exclusion. Further studies are needed to investigate if true stem cells reside in this population. Capsid-modified oncolytic viruses Ad5.pK7-∆24 and Ad5/3-∆24 seemed effective in the killing of CD44+CD24−/low cells in vitro and in vivo, including the Hoechst excluding subpopulation. When established CD44+CD24−/low–derived tumors were treated, tumor growth could be abrogated, but complete tumor eradication was not seen in most mice. This suggests that CD44+CD24−/low–derived tumors are relatively treatment resistant. Nevertheless, the same is true for many cell line–derived tumors and suggested reasons include intratumoral complexities such as stromal areas, hyperbaric, hypoxic, and necrotic areas.11,15,19,30–37 As an alternative hypothesis, the lack of complete eradication of CD44+CD24−/low–derived tumors might be explained in part by killing of only differentiated tumor cells while CD44+CD24−/low would remain resistant and their proportion would decrease because of differentiation into sensitive tumor cells. However, the lack of tumor formation following an injection of infected CD44+CD24−/low cells (Figure 4), taken together with the in vitro oncolysis data (Figure 3), seems to suggest a possible replication in and lysis of CD44+CD24−/low cells by oncolytic adenovirus in vivo as well. When CD44+CD24−/low cells were injected into mice, tumors formed rapidly. Interestingly, even though 100% CD44+CD24−/low cells were injected, the proportion of these cells returned to 11% after a tumor had developed (in untreated mice). This supports the notion of asymmetric cell division, a feature associated with stem cells. Further, treatment with Ad5.pK7-∆24 and Ad5/3-∆24 reduced the proportion of CD44+CD24−/low and Hoechst excluding cells in those tumors, suggesting activity against cells suspected as initiating tumors, metastases or relapse. In conclusion, much further work is required to understand and analyze these elusive cells, which may be important with regard to curing advanced cancers. Ultimately, clinical trials will be needed to study if Ad5.pK7-∆24 and Ad5/3-∆24 can enhance the survival of breast cancer patients. However, promising preclinical data has been obtained using both viruses in breast cancer cell lines, clinical specimens, and orthotopic animal models of both locally advanced and metastatic breast cancer.22,23 Therefore, these viruses may be effective in the killing of both “differentiated” and “tumor initiating” breast cancer cells, which might in turn be useful for improving patient survival. Further, the favorable (preliminary) biodistribution, and toxicity data set the stage for clinical translation.23 Because in vitro work and animal models cannot fully recreate the human environment, clinical studies will be needed to ultimately demonstrate the safety of these agents with regard to normal human stem cells and other normal tissues. 2091
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Finally, improved understanding of cancer stem cells, if indeed they exist, may have tremendous relevance for how cancer should be treated. It is ominous that cancer stem cells have been reported resistant to almost all anti-tumor approaches that have already been established for the treatment of metastatic disease such as ionizing radiation, hormonal therapy, chemotherapy, and small molecular inhibitors.5,7,38,39 Nevertheless, it is promising that our results suggest that they may be susceptible to capsid-modified oncolytic adenoviruses.
tumors in four mice in each group, i.e., totally 40 tumors in 20 mice. The quantity of CD44+CD24−/low cells available limited the number of mice that could be included in the experiments. Mice were also injected with 1 mg/kg Estradurin (Pfizer, New York, NY) every 3 weeks throughout the experiment. Tumors were measured with a caliper two to three times/ week. Intratumoral injections were performed with 3 × 109 viral particles of indicated viruses at time points shown by arrows in figures. Tumor volume was calculated: length × width2 × 0.5. The Provincial Government of Southern Finland approved the animal experiments.
Materials and methods
9.1; SAS, Cary, NC) was used for comparison of tumor sizes. Data was logtransformed for normality. The effects of the treatment group, time and the interaction between the treatment group time were evaluated by F tests. Curvature in the growth curves was tested by a quadratic term for time. A priori planned comparisons of differences in the predicted treatment means of all groups to mock were computed by Tukey–Kramer adjusted two-sided t-statistics averaged over all time points.
Isolation of breast cancer stem cells. Pleural effusions were obtained
(with ethics committee approval and after obtaining an informed consent) directly from thoracocentesis and washed with Dulbecco’s modified Eagle’s medium-F12 supplemented with 10 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 µg/ml insulin, and 0.4% bovine serum albumin (all from Sigma, St. Louis, MO). JIMT-1 cells were cultured according to normal conditions. Cells were sorted with fluorescein isothiocyanate–labeled anti-CD44 and phycoerythrin-labeled anti-CD24 antibodies (BD Pharmingen, Franklin Lakes, NJ), which were collected with fluorescein isothiocyanate- and phycoerythrin-conjugated magnetic beads, respectively (Miltenyi Biotech, Bergisch Gladbach, Germany). The sorted cell populations were confirmed to be CD24 negative and CD44 positive by flow cytometry. Both unsorted and CD44+CD24−/low living cell populations were stained with Hoechst 33342 (5 µg/ml; Sigma, St. Louis, MO) at 37 °C, mounted on glass slides and viewed under a fluorescence microscope. Expression of stem cell markers. Messenger RNA was isolated using
RNAeasyTM (Qiagen, Hilden, Germany). The following primers were used for real-time-PCR; oct4 forward 5′-CGCACCACTGGCATTG TCAT-3′, reverse 5′-TTCTCCTTGATGTCACGCAC-3′, sox2 forward 5′-GGCAGCTACGCATGATGCAGGAGC-3′, reverse 5′-CTGGTCATG GAGTTGTACTGCACG-3′ β-actin forward 5′-CGAGGCCCAGAGCA AGACA-3′, reverse 5′-CACAGCTTCTCCTTAATGTCACG-3′. Immu nofluorescence staining of paraformaldehyde-fixed CD44+CD24−/low cells was performed with Oct3/4 and CD44 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; BD Pharmingen, Franklin Lakes, NJ). Oncolytic adenoviruses. Viruses used have been described in detail previ-
ously: Ad5-∆24E3+ (ref. 10), Ad5/3-∆24 (ref. 20), Ad5-∆24RGD (ref. 21), Ad5.pk7-∆24 (ref. 23). Positive and negative controls were wild-type adenovirus Ad300wt (ATCC VR-5) and replication deficient Ad5luc1 (ref. 21). Viruses were produced, purified, validated, and quality controlled according to standard methods.11 Cytotoxicity assays. Cell viability was measured 5–14 days after tripli-
cate infection utilizing CellTiter 96 AQueous One Solution cell proliferation Assay (Promega, Madison, WI), which measures mitochondrial activity as a surrogate for the number of live cells. Assays were performed 8 days (PL1, PL4, and JIMT-1), 14 days (PL8), and 12 days (PL11 and PL13) after virus infection. Samples used in cytotoxicity assays contained 100% CD44+CD24−/low cells because virus infection was performed after sorting, except PL1, which was analyzed without sorting. The complete PL1 sample was used for infection and therefore the proportion of CD44+CD24−/low cell could not be analyzed. The total number and proportion of viable cells in patient sample PL11 was also studied with trypan blue staining (Invitrogen, Carlsbad, CA) 8 days after infection. In vivo studies. 2 × 106 sorted or unsorted patient pleural effusion or JIMT-1
cells were injected together with Matrigel (BD Pharmingen, Franklin Lakes, NJ) into the uppermost mammary fat pads of non-obese diabetic/severe combined immunodeficient mice. Figure 4a: N = 14 tumors in seven mice. Figure 4b: N = 10 tumors in five mice. Figure 5: N = 8
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Statistics. The repeated measures growth model in PROC MIXED (SAS
Acknowledgments We thank Eerika Karli (University of Helsinki and Helsinki University Central Hospital) and Aila Karioja-Kallio (University of Helsinki and Helsinki University Central Hospital) for their superb technical assistance. This study was supported by the European Union framework project 6 THERADPOX and APOTHERAPY, Helsinki University Central Hospital Research Funds, Sigrid Juselius Foundation, Academy of Finland, Emil Aaltonen Foundation, Finnish Cancer Society, University of Helsinki, the Finnish Oncology Association, Helsinki Graduate School in Biotechnology and Molecular Biology and the Finnish Cultural Foundation. A.H. is K. Albin Johansson Research Professor of the Foundation for the Finnish Cancer Institute. No conflicts of interest apply.
Supplementary Material Figure S1. Additional samples analyzed with FACS as described in legend for Figure 1. Figure S2. Oncolysis of CD44+CD24−/low cells from additional samples was analyzed as in Figure 3.
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