Genetically engineered stem cells efficiently deliver therapeutic proteins to cancer and other sites of inflammation. However, a major advantage would be realized if tumor-trafficking stem cells that have not been genetically modified exhibit an inherent antitumor effect, thus circumventing the necessity of the expression of exogenous genes by the cells. We transplanted Fisher 344 rat–derived mammary adenocarcinoma cells (Mat B III) orthotopically into syngeneic F344 rats with an intact immune system. Rat umbilical cord matrix stem (rUCMS) cells derived from Wharton's jelly were then administered intratumoral (i.t) or i.v. 4 days later. The tumor attenuation effect was significantly evident starting from day 14 in i.v. and i.t. rUCMS cell–transplanted rats compared with sham-transplanted rats. In addition, unmodified rUCMS cell–transplanted rats showed complete regression of tumors to undetectable levels by 34 to 38 days with no evidence of metastasis or recurrence 100 days post–tumor cell inoculation. Dye-loaded rUCMS cells were identified within tumors only 4 days after their i.v. transplantation. In vitro colony assays with rUCMS cells as feeder layers markedly reduced Mat B III colony size and number. Growth attenuation of Mat B III cells exposed to either rUCMS cells directly or to the conditioned medium derived from rUCMS cells was associated with apoptosis indicators, including increased activated caspase-3. In addition, rUCMS cells cocultured with Mat B III cells had a dose-dependent antiproliferative effect on Mat B III cells. These findings suggest that unmodified human UCMS cells could be used for targeted cytotherapy for breast cancer. [Cancer Res 2009;69(5):1815–20]
- rat mammary carcinoma
- umbilical cord matrix stem cells
It has been shown convincingly that stem cells have the ability to traffic to tumors, and signals that mediate this effect seem to be similar or identical to those that mediate recruitment of stromal or defensive cells in tumors ( 1– 3). There are also a number of reports showing that genetically engineered stem cells efficiently deliver therapeutic proteins to cancer and other sites of inflammation ( 2– 8). Stem cells we have isolated from the Wharton's jelly of umbilical cord, termed “umbilical cord matrix stem” (UCMS) cells also exhibit ability to traffic selectively to tumors ( 3, 9). When these cells are engineered to secrete a cytokine, IFN-β, and are administered i.v., they can attenuate metastatic breast cancer in a severe combined immunodeficient (SCID) mouse model ( 3). In the current study, we used a syngeneic F344 rat mammary adenocarcinoma model with an intact immune system because it more closely models clinical human cancer. We isolated UCMS cells from the Wharton's jelly of rat umbilical cord, termed rat UCMS (rUCMS) cells, to use as potential delivery vehicles for targeted gene therapy. As a necessary preliminary step, we evaluated the effect of unengineered rUCMS cells on Matt B III cancer cells in vitro and in vivo. To our surprise, the stem cells themselves mediated a profound antitumor effect in both in vivo and in vitro. Here, we describe these findings and investigate several potential mechanisms involved in the antitumor effect of rUCMS cells.
Materials and Methods
Antibodies against caspase-3, cleaved caspase-3, extracellular signal-regulated kinase (ERK)1/2, phospho ERK1/2, p38, phospho p38, and poly(ADP)ribose polymerase were purchased from Cell Signaling Technology. Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), c-Jun-NH2-kinase (JNK), and phospho JNK were purchased from Santa Cruz Biotechnology. Enhanced chemiluminescence (Amersham) anti-rabbit IgG, mouse IgG horseradish peroxidase (HRP) antibody were purchased from GE Healthcare.
rUCMS cells were harvested from E19 pregnant rats. The method to isolate and culture rUCMS cells was previously described ( 9). rUCMS cells were maintained in low-serum (defined) medium, containing the following mixture per 100 mL: 57 mL low-glucose DMEM (Invitrogen), 37 mL MCBD 201 (Sigma), 2 mL fetal bovine serum (FBS; Atlanta Biologicals, Inc.), 1 mL of 100× insulin-transferrin-selenium-X (Invitrogen); 1 mL of 0.15 g/mL AlbuMax1 (Invitrogen), 1 mL of 100× Pen/Strep (Invitrogen), 10 nmol/L dexamethasone (Sigma), 100 μmol/L ascorbic acid 2-phosphate (Sigma), 10 ng/mL epidermal growth factor (R&D systems), and 10 ng/mL platelet derived growth factor-BB (R&D systems). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
The rat mammary adenocarcinoma cell line, Mat B III, was maintained in McCoy's 5A modified medium (Invitrogen) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
In vivo Studies
All experiments were carried out using protocols approval by the Institutional Animal Care and Use Committee (IACUC).
Experimental design I. Female 4- to 5-mo-old F344 rats were obtained from Charles River laboratories. All rats were housed in a clean facility and held for 1 wk to acclimatize. On day 0, 1 × 105 Mat B III tumor cells suspended in 100 μL of PBS were implanted orthotopically into the abdominal mammary fat pad under isoflurane anesthesia. All rats were randomized into four treatment groups: 1, intratumoral (i.t.) PBS injection (n = 5); 2, i.t. rUCMS cells (0.5 × 106) transplant (n = 5); 3, i.t. rUCMS cells (1 × 106) transplant (n = 10); and 4, i.v. rUCMS cells (3 × 106) transplant (n = 5). On day 4 post–tumor cell transplantation, rUCMS cells (0.5 × 106 or 1 × 106 cells) suspended in 100 μL of PBS were injected i.t. For i.v. stem cell treatment, 1.5 × 106 rUCMS cells suspended in 200 μL of PBS were injected via tail vein on days 4 and 6 post–tumor cell transplantation. Tumor-bearing rats given i.t. 100 μL of PBS on day 4 served as control rats. Tumors became palpable starting from day 7; tumors were measured every 3 to 4 d with calipers under isoflurane anesthesia. Results from a pilot study showed that tumors reach 2 ± 0.2 cm (greatest diameter) by the end of day 18. All animals were sacrificed on day 18, and tissues including lung, liver, spleen, kidney, and tumor were collected for further analysis.
Experimental design II. After Mat B III cell transplantation, all rats were randomized into three treatment groups: 1, i.t. PBS injection (n = 6); 2, i.t. rUCMS cells (1 × 106) transplant (n = 6); and 3, i.v. rUCMS cells (3 × 106) transplant (n = 6). On day 4, 1 × 106 rUCMS cells per rat were injected i.t.; 1.5 × 106 UCMS cells per rat were injected i.v. via tail vein on day 4 and repeated 2 d later. Tumors were measured every 3 to 4 d starting from day 8 until the size of the tumor reached 2 ± 0.2 cm diameter. Animals with tumors of 2 ± 0.2 cm diameter were sacrificed and tumors were collected for further analysis.
Identification of rUCMS Cells in Rat Tissues
In a separate experiment performed to evaluate the trafficking of transplanted rUCMS cells at various time points after transplant, the fluorescent dye SP-DiI (Molecular Probes) was dissolved in DMSO at a concentration of 5 mg/mL. SP-DiI dye was added to culture medium to a final concentration of 10 μg/mL and added to rUCMS cells in a T-75 flask for 12 h. Then, cells were washed with 1×PBS and incubated with dye-free medium for 4 h, after which they were used for i.t. or i.v. transplantation. All tumor transplanted rats were randomized into 3 treatment groups: 1, i.t. PBS injection; 2, i.t. rUCMS cells (1 × 106) transplant; and 3, i.v. rUCMS cells (3 × 106) transplant. On day 4, 1 × 106 SP-DiI loaded rUCMS cells were injected i.t. and 3 × 106 SP-DiI–loaded rUCMS cells were injected i.v. via tail vein. On days 8, 14, 18, and 22 rats were sacrificed, and tissues including tumor, lung, spleen, liver, and kidney were snap-frozen in liquid nitrogen for histologic analysis. Tissues were sectioned on a cryostat at 7 to 8 μm and random tissue sections were counter stained with 4′,6-diamidino-2-phenylindole nuclear stain and observed under epifluorescence microscopy (Nikon Eclipse; Boyce Scientific, Inc.). Images were captured using a Roper Cool Snap ES camera and Metamorph 7 software.
Tissue were fixed in 10% buffered neutral formalin, routinely processed, embedded in paraffin, sectioned at 5 μm, and stained with H&E for microscopic examination. The slides were later interpreted by a board-certified pathologist (G.A.).
Colony Formation Assay
rUCMS cells from passage numbers ranging from 10 through 14 were seeded at a density of 5 × 103 cells per well in a 6-well tissue culture plate containing defined medium supplemented with 5% FBS. When rUCMS cells were grown to ∼30% confluency, 1 mL 0.9% agar (Sea Plaque agarose; Cambrex Bio Science Rockland, Inc.) in 5% FBS-defined medium was poured into the dish (bottom layer). Mat B III cells (2 × 103 cells per well) were suspended in 1 mL of 5% FBS-defined medium containing 0.5% agarose and plated on top of the bottom agarose layer. The cells were incubated at 37°C with 5% CO2 for 10 d for growth of colonies. Colonies >5 × 104 μm2 were counted by an automated phase contrast analyzing microscope equipped with Micro Suite Analysis Suite (Olympus CKX41).
[3H] Thymidine Uptake Assay
The [3H] thymidine uptake assay was carried out as described previously ( 10). In all [3H] thymidine incorporation experiments, rUCMS cells (5 × 103 or 1 × 104 per well) were plated in 24-well culture plates and were cultured in CO2 incubator for overnight. Mat B III cells at a density of 1.5 × 105 cells per well were added to the rUCMS cells grown in 24-well plates, incubated for 24 h, treated as indicated in the figure legends, and pulsed for the last 4 h of the treatment time with 1.0 μCi [3H] thymidine per well. Mat B III cells in suspension culture were collected, and the free-[3H] thymidine in the medium was washed away with PBS. The cell-incorporated [3H] thymidine was solubilized by adding 0.2 mol/L NaOH and counted by the Packard liquid scintillation counter Tri-Carb 2100TR (Perkin-Elmer Life Science).
Protein Extraction and Western Blotting
Indirect coculture of rUCMS and Mat B III cells. Mat B III tumor cells were grown in 12-well culture plates with defined medium containing 5% FBS. After the cells reached ∼90% confluency, the medium was replaced with 1:1 ratio of defined medium and conditioned medium (CM) obtained from a 72-h culture of rUCMS cells. After incubation, cells were collected at various time points as indicated in the figure legends. Mat B III cells were collected and pelleted for further analysis.
Direct coculture of rUCMS and Mat B III cells. The rUCMS cells (1 × 104 or 5 × 104 per well) were plated in a 12-well culture plate and were cultured in CO2 incubator overnight. Mat B III cells at a density of 3 × 105 cells per well were added to the rUCMS cells grown in 12-well plates and further incubated for 48 h. Mat B III cells in suspension were collected and pelleted for further analysis.
Mat B III cell pellets were lysed with cell lysis buffer [1% Triton, 0.1%SDS, 0.25M sucrose, 1 mmol/L EDTA, 30 mmol/L Tris-HCl (pH 8.0), and 7× complete mini EDTA-free stock solution (Roche Diagnostics)] at ratio of 1:7 followed by sonication and boiling. The cell lysates were mixed with 1/4 volume of 5× SDS-PAGE sample buffer. Proteins were separated with 8% to 12% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare). The membrane was probed with a specific primary antibody at dilution of 1:1,000 to 1: 4,000, followed by washing and probing with anti-rabbit HRP-antibody at dilution of 1:2,000. The specific protein bands were visualized by a Kodak image station using the Super Signal kit (Pierce Biotechnology). Western blots were quantified by image analysis using Scion Image software.
All values are expressed as means ± SE. The differences in the tumor size and weight after PBS or i.t. and i.v. stem cell transplants, the differences in Mat B III colony sizes in different culture conditions, and the differences in the [H3] thymidine uptake after coculturing Mat B III cells with different number of rUCMS cells were compared using Student's t tests. Statistical significance was considered if P value was <0.05.
Characterization rUCMS cells. The rUCMS cells were derived from Wharton's jelly of rat umbilical cord and propagated using previously described protocols ( 9). Morphologically, these cells resemble bone marrow mesenchymal stem cells (MSC) and are plastic adherent (Supplementary Fig. S1A). We tested rUCMS cells for the expression of MSC markers. Flow cytometry analysis showed that rUCMS cells express MSC markers CD73 and CD90 (Supplementary Fig. S1B). Real-time reverse transcription-PCR analysis showed no expression of the embryonic stem cell markers OCT4, Nanog, SOX2, or ESG2 (data not shown).
rUCMS cells significantly attenuate rat mammary adenocarcinoma growth. We tested the ability of unengineered (naïve) rUCMS cells to home to tumor areas and analyzed their effect on tumor growth. Histologic analysis revealed that engraftment of rUCMS cells was detected in close proximity to or within tumor tissues of tumor-bearing rats that received SP-DiI–labeled rUCMS either i.t. or i.v. ( Fig. 1C ). No SP-DiI–labeled rUCMS cells were found in other tissues, such as lung, liver, spleen, or kidney on day 4 (data not shown). As shown in Fig. 1A, i.t. and i.v. transplantation of rUCMS cells significantly attenuated tumor growth starting at day 14 to day 18 compared with PBS-i.t. (sham) rats. In addition, there was a significant dose-dependent tumor attenuation effect in rats injected i.t. with 1 × 106 rUCMS cells compared with rats injected with 0.5 × 106 rUCMS cells ( Fig. 1A). All rats were sacrificed on day 18 as tumors reached the size of 2 ± 0.2 cm diameter in sham-transplanted rats. The tumor weights measured on day 18 showed a significant difference between i.t. or i.v. rUCMS-transplanted rats compared with sham-transplanted rats. There was also a significant dose-dependent difference in tumor weights between rats receiving i.t. 1 × 106 rUCMS cells and those receiving i.t. 0.5 × 106 rUCMS cells ( Fig. 1B).
We further tested whether there would be any recurrence of tumors after a certain period of time. As shown in Fig. 2A , the i.t. and i.v. rUCMS cell–transplanted rats showed a significant attenuation of tumor growth compared with sham-transplanted rats (n = 6; sacrificed on day 18 after tumors reached 2 ± 2 cm diameter). The tumors regressed and were not palpable by the end of day 34 in i.v. transplanted rats (n = 4), and by day 38 in i.t. transplanted rats (n = 6). Two i.v. transplanted rats were sacrificed on day 22 as the tumors did not regress (in accordance with IACUC mandated protocol). We speculate that it is due to injection of an inadequate number of viable rUCMS cells i.v. (as sometimes happens via this route); note that the data in Fig. 1A indicates the rUCMS cell dose-dependent attenuation of tumor size. Figure 2B shows a representative of the tumor in a sham-transplanted rat (left) and an i.v. rUCMS cell–transplanted rat (right) showing complete regression of the tumor in the i.v. rUCMS-transplanted rat. Histopathologic analysis of tumors revealed the tumors in sham-transplanted rats contain mostly anaplastic tumor cells with a very thin layer of granulation tissue and small numbers of lymphocytes, few macrophages, and neutrophils, whereas in i.t. and i.v. rUCMS cell–transplanted rats, there are few islands of neoplastic cells with a thick granulation tissue and infiltrated by moderate to large numbers of lymphocytes often mixed with plasma cells ( Table 1 ).
rUCMS cells inhibit contact-independent and contact-dependent growth of Mat B III cell line. The colony formation of Mat B III cells in soft agar was significantly attenuated when rUCMS cells ( Fig. 3A, 1 ) were cocultured in the bottom of culture dish. However, control rat skin fibroblasts ( Fig. 3A, 2) obtained from F344 newborn pups did not affect the colony formation. The results from the colony assay were summarized in Fig. 3B and showed a significant decrease in the number of Mat B III colonies when cocultured with rUCMS cells compared with fibroblasts. In Fig. 3C, Mat B III cells cocultured with a small number of rUCMS cells (1 × 104) markedly inhibited [3H] thymidine uptake by Mat B III cells (1.5 × 105). We further investigated the effect of rUCMS cells on the growth and survival of Mat B III cells. The Mat B III cells were cocultured with either the medium conditioned with rUCMS cells (CM) or unconditioned medium for different times. As shown in Fig. 4A , ERK1/2 phosphorylation (top) progressively decreased over time, whereas p-38 phosphorylation was increased at the 24-hour time point. To study the direct interaction of rUCMS cells on cell growth or apoptosis, small numbers of rUCMS cells were cocultured with Mat BIII cells (1 of 30 or 1 of 6) for 72 hours. Western blot results showed that coculture with rUCMS cells significantly decreased the phosphorylation of AKT and attenuated the phosphorylation of ERK1/2 and decreased levels of procaspase 3 but significantly increased cleaved caspase-3 in Mat B III cells in a dose-dependent manner ( Fig. 4B).
The present study shows for the first time the complete elimination, with no evidence of recurrence, of rapidly growing syngeneic rat mammary adenocarcinoma cells by unmodified, naïve rUCMS cells. This effect was observed in most tumor-bearing rats transplanted systemically with rUCMS cells, and in all of those rats with stem cells transplanted into the tumor. In addition, we describe here the following important new findings: (a) rUCMS cells administered i.v. “home” to the orthotopic tumors only 4 days later; (b) orthotopic Mat B III mammary carcinoma size decreases over time after rUCMS cells are administered i.v. or i.t.; (c) there is a dose-dependent attenuation of tumors in rats transplanted with different doses of i.t. rUCMS cells; (d) in vitro three-dimensional colony formation of Mat B III mammary carcinoma cells is markedly attenuated when rUCMS cells but not fibroblasts are present as monolayers below the cultures; (e) Mat B III cells incubated with CM derived from rUCMS cells show decreased phosphorylation of ERK1/2 and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK); (f) Mat B III cells cocultured with rUCMS cells show decreased phosphorylation of ERK1/2 and AKT, and decreased procaspase 3 along with increased cleaved caspase-3 levels; and (g) a [3H] thymidine uptake assay shows potent inhibition of Mat B III cell proliferation in the presence of rUCMS cells in a dose-dependent manner.
The homing of stem cells to tumors and other areas of inflammation is well-established ( 1– 7, 11). Here, we administered i.v. rUCMS cells via the tail vein and observed that they could find their way into tumors only 4 days later. The homing ability of stem cells seems to be mediated by chemokines and growth factors ( 12) secreted by the tumors or their associated stroma ( 13, 14).
The homing ability of stem cells has previously been exploited for drug delivery and targeted gene delivery ( 1– 8). The ability of unengineered UCMS cells to completely eliminate mammary adenocarcinomas is a distinct advantage because any manipulation causing the cells to express an exogenous gene could alter them in some way that would potentially make them less safe as transplantable cells. Our findings are in agreement with a recent report showing that human MSCs that have not been genetically modified can attenuate Kaposi's sarcoma in vivo by >50% after systemic administration, although in that report, the stem cells were given in great numbers and did not completely eradicate the tumors ( 15). The antitumor effect shown in our study may be due to the combined effect of a potent antiproliferative effect as well as a proapoptotic effect mediated by the naïve rUCMS cells on the Mat B III cells. Because tumors were completely abolished with no evidence of recurrence in rUCMS transplanted animals, the cancer initiating (stem cell) population seems to have been eliminated.
Previous in vivo studies have shown that non–stem cells do not exhibit homing and/or the antitumor effect. For example, Nakamizo and colleagues ( 2) showed that rat fibroblasts administered into the ipsilateral carotid artery of mice with U87 glioma showed nonspecific migration to tumors. Khakoo and colleagues ( 15) showed no effect of human umbilical vein endothelial cells on Kaposi sarcoma tumor growth. In the present study, in vitro colony assay results showed no effect of rat fibroblasts on Mat B III colony growth.
Evidence indicates a strong nonimmune component of the antitumor effect of UCMS cells. For example, previous studies from our laboratory showed that unengineered human UCMS cells transplanted i.v. showed a tendency to attenuate metastatic human breast cancer cells in SCID mice; however, the effect was not significant ( 3). Here, the dramatic reduction in the Mat B III tumor colony size and number when rUCMS cells (but not rat fibroblasts) were used as a feeder layer must have been mediated by diffusible factors secreted by the rUCMS cells, as there was no direct contact between the two cell types. Human UCMS cells secrete significant amounts of cytokines that are associated with antitumor effects ( 16). Results from our laboratory using a rat common cytokine PCR array showed rUCMS cells alone express high levels of antitumor agents and even increased expression when cocultured with Mat B III tumor cells (data not shown).
Attenuation of tumor cell growth by rUCMS cells seems to have both contact-independent and contact-dependent components. As shown in Fig. 4A, the CM from rUCMS cells stimulates phosphorylation of p38 and decreases phosphorylation of ERK1/2, suggesting contact-independent inhibition of Mat B III tumor cell growth. Interestingly, Khakoo and colleagues ( 15) showed that the MSCs in vitro could only mediate their effect on Kaposi sarcoma by contact with the tumor cells. Here, we showed that in a [3H] thymidine uptake assay ( Fig. 3B), rUCMS cocultured with Mat B III cells significantly attenuate Mat B III cell proliferation. Because the number of rUCMS cells was significantly smaller than Mat B III, this rUCMS cell–dependent effect is not strictly contact mediated; there is likely some factor(s) independent of cell-to-cell contact that is involved. Our coculture results suggest that the rUCMS cell–dependent growth attenuation is due at least in part to the decreased phosphorylation of Akt and ERK1/2, and to decreased procaspase-3 and increased cleaved caspase-3 levels ( Fig. 4B). The chemical nature of the factor or factors produced by rUCMS cells that attenuated the growth of Mat B III cells is unclear. However, aforementioned results suggest that the rUCMS cell factor may be a relatively stable, diffusible small molecule that can attenuate phosphorylation of MAPK and Akt and stimulate apoptosis. However, identification of this factor awaits another study.
UCMS cells (genetically modified or not) have many potential advantages for cytotherapy. They can be harvested in large numbers in a short amount of time, harvested noninvasively, and are noncontroversial ( 9). Porcine UCMS cells elicit only minimal immune responses as shown by one-way mixed lymphocyte reaction; in fact, completely MHC-mismatched UCMS cells caused only a minimal immune response after a single transplantation ( 17, 18). The findings described here have important implications for patients with breast cancer and other types of cancer. Umbilical cord matrix stem cells may represent a new therapeutic modality for the treatment of cancer. Ongoing research is directed toward further elucidation of mechanisms that may be involved, including potential immunomodulation of the host.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Kansas State University Terry C. Johnson Center for Basic Cancer Research, Kansas State University Targeted Excellence, Kansas State Legislative Appropriation, and Kansas State University College of Veterinary Medicine SMIEL grant.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Mal Hoover and Rachel Salmans for their technical support.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received July 18, 2008.
- Revision received December 23, 2008.
- Accepted December 31, 2008.
- ©2009 American Association for Cancer Research.