Anti-OX40 monoclonal antibody therapy in combination with radiotherapy results in therapeutic antitumor immunity to murine lung cancer

The therapeutic effect of agonistic anti-OX40 (CD134) monoclonal antibody (mAb) in combination with radiotherapy was evaluated in a murine lung cancer model. After intradermal transplantation of ovalbumin (OVA)-transfected Lewis lung carcinoma, C57BL/6 mice were irradiated locally with a single dose of 20 Gy in combination with an intratumoral injection of anti-OX40 mAb at 50 µg on day 4 after transplantation, which is when the major axis of the inoculated tumor reached a diameter of 7–9 mm. On days 8, 11, and 14, the tumor-bearing mice were further treated with the same dose of anti-OX40 mAb. Anti-OX40 mAb in combination with radiotherapy prolonged survival and provided greater efficacy than either single treatment against well-established tumors. An in vivo depletion study suggested that therapeutic immunity was mainly CD8⁺ T-cell dependent. OX40⁺CD8⁺ T cells were augmented in draining lymph nodes obtained from irradiated mice compared with those from non-irradiated mice. OVA-major histocompatibility complex tetramer⁺ CD8⁺ T cells had been strongly recruited to the draining lymph nodes obtained from mice treated with anti-OX40 mAb in combination with radiotherapy, and strong antigen-specific cytotoxicity was confirmed by a ⁵¹Cr-release assay. Moreover, a tumor-rechallenge model indicated that this combination therapy induced durable tumor immunity. Thus, anti-OX40 mAb in combination with radiotherapy may potentially help the management of patients with lung cancer. ( Cancer Sci 2008; 99: 361–367)     

Efficient Gene Transduction by RGB-fiber Modified Recombinant Adenovirus into Dendritic Cells

Dendritic cells (DC) are important antigen-presenting cells in the development of an anti-tumor T cell response. To extend the range of current immuno/gene therapies, we tested luciferase-expressing RGD-adenovirus (Ad) (Ad5lucRGD)-mediated transduction into DC. Phenotypically characterized DC were generated from peripheral blood CD14⁺ cells by incubation with granulocyte-macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor a. On the 7th day of culture, the cells became mature DC with a CD1a⁺, CD11c⁺, CD80⁺, CD83⁺, CD86⁺, human leukocyte antigen (HLA)-DR⁺, CD14⁻ phenotype. The expression of (α,β₃ integrin was enhanced on day 3 and returned to the basal level on day 7. We then compared the transduction efficiency of an AdSlucRGD system to that using conventional Ad, in cells harvested on days 1, 3 and 7 of culture. Luciferase activity was negligible in AdCMVLuc, but remarkable in cells processed with Ad5lucRGD. Activity was maximal in cells that had been cultured for 3 days. Recombinant Ad5 fiber knob protein blocked AdCMVLuc- and Ad5lucRGD-mediated gene transduction by 90% and 20%, respectively. Surface markers and cytokine production were not affected by Ad5lucRGD-mediated transduction.     

Sequential Involvement of Two Distinct CD4+ Regulatory T Cells during the Course of Transplantable Tumor Growth and Protection from 3–Methylcholanthrene-induced Tumorigenesis by CD25–depletion

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4⁺CD25⁺ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4⁺CD25⁻ T cells downregulated the tumor rejection response in the late stage. Both CD4⁺CD25⁺ and putative CD4⁺CD25-T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3–methylcholanthrene (3–MC) inoculation retarded tumor occurrence and prolonged survival.     

The Roles of CD8+ and CD4+ Cells in Tumor Rejection

In vivo administrations of anti-Lyt-2.2 (CDS) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8⁺ cells amd CD4⁺ cells, respectively. The relative potencies of CD8⁺ cells and CD4⁺ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8⁺ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL♂1, and a plasmacytoma BALBMOPC-70A in CB6F₁ mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4⁺ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F₁UV+^˚l sarcoma by CB6F₁ mice, synergy of CDS⁺ and CD4⁺ cells was necessary. Blocking of UV+^˚ 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CDS) mAb, indicating the involvement of CD4⁺ cells in only the initial phase of rejection.     

Expression of Mucin-associated Sulfo-Lea Carbohydrate Epitopes on Human Colon Carcinoma Cells

The level of sulfo-Le^a (SO₃-3Galβ1-3(Fucα1-4)GlcNAc) epitope recognized by monoclonal antibody (mAb) 91.9H in hepatic metastasis of colon carcinoma is known to be lower than at the primary sites. We examined 19 human colon carcinoma cell lines for their production of this epitope. Sixteen cell lines were found to produce high M _r components that metabolically incorporated [³⁵S]sulfate and were resistant to heparitinase I and chondroitinase ABC, and 8 of them were reactive with mAb 91.9H as shown by western blotting analysis. These were all of the 4 cell lines derived from well differentiated primary tumors (HCCP-2998, LS174T, GEO, and CBS), 2 of 10 cell lines (DLD-1 and HCT116) from moderately to poorly differentiated primary tumors, and 2 of 5 cell lines (SW480 and HCC-M1544) from metastases. Incubation of LS174T cells with benzyl- N -acetyl-α- d -galactosaminide abrogated the incorporation of [³⁵S]sulfate and the reactivity of mAb 91.9H with high M _r components in the cell lysates. Sodium chlorate, which inhibits the formation of 3'-phosphoadenosine 5'-phosphosulfate, also inhibited the [³⁵S]sulfate incorporation and reactivity with mAb 91.9H. These treatments did not change the incorporation of [¹⁴C]threonine into high M _r components. These results indicated that sulfo-Le^a epitopes were expressed on O -linked carbohydrate chains in sulfomucins. Immunohistochemical studies of tumor tissues in nude mice indicated that sulfo-Le^a was expressed at the site of orthotopic transplantation in the cecum. The expression appeared to be suppressed in liver metastatic foci in nude mice.     

Augmentation by Bispecific F(ab')2 Reactive with P-Glycoprotein and CD3 of Cytotoxicity of Human Effector Cells on P-Glycoprotein Positive Human Renal Cancer Cells

A bispecific F(ba')₂ was constructed that was composed of two Fab fragments, one derived from anti-CD3 monoclonal antibody (mAb) (OKT3) and the other from anti P-glycoprotein mAb (MRK 16). This bispecific F(ab')₂ enhanced the binding and cytotoxicity of human peripheral blood mononuclear cells (PBMCs) on P-glycoprotein-positive human kidney cancer cells (ADMHK/E). It had no effect on the cytotoxicity of PBMCs on P-glycoprotein-negative HK/E cells [long-term cultured HK/E (LCHK/E)]. Control F(ab')₂ composed of OKT3 or MRK16 alone did not influence the cytotoxicity of PBMCs on ADMHK/E cells. These findings suggest that the MRK16-OKT3 bispecific F(ab')₂ may be therapeutically beneficial in treatment of human multidrug-resistant cancers.     

Multipotent and Committed CD34+ Cells in Bone Marrow Transplantation

In order to study the role of CD34⁺ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34⁺ cells were also performed to evaluate their in vitro hematopoietic function. CD34⁺ cells were detectable in bone marrow cells on day 14. More than 80% of CD34⁺ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34⁺ cells. In normal bone marrow cells, CD34⁺, CD33⁺ cells amounted to about 40% of CD34⁺ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34⁺, CD33⁻ cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34⁺, CD33⁺ cells as committed progenitors and CD34⁺, CD33⁻ cells as multipotent stem cells have distinctive biological behaviors in BMT.     

Inhibition of Cell Attachment, Invasion and Metastasis of Human Carcinoma Cells by Anti-integrin β1 Subunit Antibody

We investigated the expression of β₁ integrins in human carcinoma cell lines, and the anti-metastatic and anti-invasive effects of a newly established anti-human β₁ subunit monoclonal antibody designated NCC-INT-7. All the examined carcinoma cell lines expressed β₁ integrins upon immunoblot analysis. NCC-INT-7 completely inhibited the adhesion of carcinoma cells to laminin, fibronectin, collagens and acetone-fixed tissues including lung, liver and brain. In an in vitro invasion model, NCC-INT-7 inhibited the invasion of human bladder carcinoma cell line T24 and human gastric carcinoma cell lines TMK-1, MKN-45 and MKN-74 through an artificially reconstructed basement membrane. In an in vivo nude mouse peritoneal dissemination model using MKN-45 and TMK-1, NCC-INT-7 significantly reduced the number of tumor nodules in the mesentery. In an in vivo nude mouse liver metastasis model using a serially transplantable human colonic carcinoma, COL-2–JCK, NCC-INT-7 significantly reduced the number of tumor nodules in liver. These results indicate that β₁ integrins play an important role in the tissue attachment, migration, invasion and metastasis of human carcinoma cells, and that this new monoclonal antibody is useful for studies aimed at prevention of metastasis.     

Bispecific Antibody-directed Antitumor Activity of Human CD4+ Helper/Killer T Cells Induced by Anti–CD3 Monoclonal Antibody plus Interleukin 2

Freshly isolated human CD4⁺ T cells can not respond to recombinant interlenkin 2 (rIL–2) because of their lack of p75 IL–2 receptor expression. However, we succeeded In inducing a marked proliferation of purified CD4⁺ T cells by activation with rIL–2 plus anti–CD3 monoclonal antibody (mAb) cross–linked to a plastic plate. The proliferated CD4⁺ T cells produced a significant amount of IL–2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4⁺ T cells activated with anti–CD3 mAb plus rIL–2 revealed a strong cytotoxic activity against Fc receptor (FcR)–positive tumor cells in the presence of anti–CD3 mAb. Moreover, the CD4⁺ T cells could lyse FcR–negative glioma cells by targeting with bispecific mAb containing anti–CD3 mAb and anti–glioma mAb. Thus, we demonstrated that rIL–2 and immobilized anti–CD3 mAb allowed the rapid generation of human CD4⁺ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Expression of the SART-1 Antigens in Uterine Cancers

We recently reported that the SART-1 gene, encoding the SART-1₂₅₉ tumor antigen which is recognized by HLA-A26-restricted cytotoxic T lymphocytes (CTLs), is expressed in the cytosol of squamous cell carcinomas and adenocarcinomas. The present study deals with the expression of SART-1₂₅₉ and SART-1₈₀₀ antigens in uterine cancers. The SART-1₂₅₉ antigen was detected in the cytosol fraction of 4 of 8 uterine cancer cell lines, 24 of 74 (32%) uterine cancer tissues, 0 of 7 uterine myomas, and 0 of 5 non-tumorous uterine tissues. The SART-1₈₀₀ antigen was expressed in the nuclear fraction of all the uterine cancer cell lines, 41 of 74 (55%) uterine cancer tissues, 0 of 7 myomas, and 3 of 5 non-tumorous uterine tissues. The SART-1₂₅₉⁺ uterine cancer cells were recognized by HLA-A24 restricted and SART-1 specific CTLs. Therefore, SART-1₂₅₉ antigen could be an appropriate vaccine candidate for a relatively large number of uterine cancer patients.     

Effects of anthracycline derivatives on hepatic neoplastic nodules of Lewis lung carcinoma and colon adenocarcinoma 26

Five anthracycline derivatives, i.e. doxorubicin, epirubicin, pirarubicin, aclarubicin and a new fluorinated anthracycline derivative (ME2303), were tested for antitumour activity in mice with hepatic neoplastic nodules of Lewis lung carcinoma and colon adenocarcinoma 26. Intravenous administrations of pirarubicin and ME2303 on day 4 or days 4, 8 and 12 in mice with hepatic neoplastic nodules of Lewis lung carcinoma rendered more than 50% of mice tumour-free over wide ranges of nontoxic doses, whereas a few mice were cured by treatment with doxorubicin and no mice were cured by treatment with epirubicin or aclarubicin. Moreover, when ME2303 was administered at 50 mg kg-1 on days 7, 11 and 15 to six mice bearing more advanced hepatic tumours, five were cured, while pirarubicin and doxorubicin never achieved cure. Furthermore, in mice with hepatic neoplastic nodules of colon adenocarcinoma 26, ME2303 also showed a marked antitumour effect compared to pirarubicin or doxorubicin. Two or three injections of ME2303 starting from day 7 conferred a greater antitumour effect than did more fractionated or single-dose regimens.     

Frameshift Mutations and a Length Polymorphism in the hMSH3 Gene and the Spectrum of Microsatellite Instability in Sporadic Colon Cancer

Mutations in the hMSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)₈ region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)₈ region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)₈ region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)₇ and (A)₆ regions in hMSH3. Thus, we confirmed that the (A)₈ region in hMSH3 is a hot spot and mutations in the (A)₇ and (A)₆ regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Analysis of CD8 T-cell response by IFNgamma ELISPOT and H-2L(d)/pRL1a tetramer assays in pRL1a multiple antigen peptide-immunized and RL male 1-bearing BALB/c and (BALB/c x C57BL/6) F(1) mice

We previously identified an H-2L^d-binding peptide pRL1a (IPGLPLSL) on RL male 1 that is predominantly recognized by cytotoxic T-lymphocytes (CTLs). MAP is a multibranched lysine core with antigenic peptides. Immunization of BALB/c mice with pRL1a MAP effectively induced pRL1a CTLs. Here, we demonstrate the presence of pRL1a-recognizing CD8⁺ T-cells in pRL1a MAP-immunized and RL male 1-bearing BALB/c and (BALB/ cxC57BL/6)F₁ mice by using IFNγ ELISPOT and H-2L^d/pRL1a tetramer assays. A few IFNγ ELISPOTs and no tetramer-positive cells were detected ex vivo in spleen cells from BALB/c mice immunized with pRL1a MAP. After a single in vitro stimulation with RL male 1, 432 and 741 IFNγ ELISPOTs/10⁵ cells were detected and tetramer-positive CD8⁺ T-cells occurred at relative frequencies of 5.7% and 30.8% in splenic CD8⁺ T-cells from mice that had been doubly and triply immunized, respectively, against pRL1a MAP. Tetramer-positive cells displayed two distinct cell populations, CD62L^low and CD62L^high. Secondary in vitro stimulation expanded CD62L^high cells more efficiently than CD62L^low cells. Furthermore, a higher frequency of IFNγ-producing and tetramer-positive CD8⁺ T-cells was detected ex vivo in RL male 1-bearing semi-allogeneic (BALB/cxC57BL/6)F₁ than in BALB/c mice on day 14 after tumor inoculation.     

Activation of Intestinal Mucosal Immunity in Tumor-bearing Mice by Lactoferrin

We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly increases CD4⁺ and CD8⁺ T cells and NK (asialoGM1⁺) cells in the blood of tumor-bearing mice and enhances anti-metastatic activity. In this paper, we document that oral administration of bLF and bLF-hydrolysate (bLFH) is associated with strong increases in CD4⁺ and CD8⁺ T, as well as asialoGM1⁺ cells in lymphoid tissues and lamina propria of the small intestine in mice, especially in tumor-bearing animals in which Co26Lu cells were implanted subcutaneously. Moreover, IgM⁺ and IgA⁺ B cells in lamina propria of the small intestine were also significantly increased by bLF and bLFH. Bovine apo-transferrin (bTF) did not exhibit such activity. In the colon, only CD8⁺ cells were significantly increased by treatment with bLF, while asialoGM1⁺ cells were significantly decreased. bLF and bLFH induced cytokines to activate T, B and asialoGM1⁺ cells. Administration of bLF and bLFH, but not bTF, increased production of interleukin-18 (IL-18), interferon-gamma (IFN-γ) and caspase-1 in the mucosa of the small intestine. Particularly high levels of IL-18 were found in the epithelial cells of the small intestine. Moreover, administration of bLF and bLFH, but not bTF, induced IFN-γ presenting cells in the small intestine. Caspase-1, which processes proIL-18 to mature IL-18, was also induced in the epithelial cells of the small intestine following treatment with bLF and bLFH, but not with bTF. These results suggest that enhanced production of IL-18 and IFN-γ and caspase-1 induction by treatment with bLF may be important for elevation of intestinal mucosal immunity.     

Enhanced Liver Metastatic Potential of Alpha-fetoprotein-producing Human Gastric Carcinoma after Carbon Tetrachloride-induced Liver Damage in Nude Mice

The liver metastatic potential of alpha-fetoprotein (AFP)-producing human gastric carcinoma (NSC-3) was examined in male, BALB/c, nude mice. Metastatic nodules in the liver were produced by intrasplenic (IS) injection of tumor cell suspension prepared by trypsinization from subcutaneous NSC-3 tumor. The serum AFP level increased exponentially after IS injection along with the growth of metastatic nodules in the liver, and a positive correlation was observed between the estimated weight of metastatic nodules and serum AFP level. To investigate the effect of liver damage by carbon tetrachloride (CCI₄) on the metastatic potential of NSC-3 cells injected intrasplenlcally, the mice were divided into 4 groups: Group 1 received IS injection of 1×10⁶ of NSC-3 cells without CCI₄, treatment; Groups 2, 3 and 4 received IS injection 7 days, 2 days and 1 day after CCI₄ treatment, respectively. All mice were killed 64 days after IS injection. The incidence of liver metastasis was 80% in Group 1, but 100% in Groups 2, 3 and 4. The mean numbers of metastatic nodules per liver were 4.2 in Group 1, 16.8 in Group 2, 18.0 in Group 3 and 44.5 in Group 4. Significant differences in the mean numbers of metastatic nodules were observed between Group 4 and the other groups. It was clearly demonstrated that the metastatic potential of AFP-producing human gastric carcinoma cells (NSC-3) is enhanced in the situation prevailing after liver parenchymal cells are damaged by CCI₄.     

Macrophage–T Cell Interaction Is Essential for the Induction of p75 Interleukin 2 (IL–2) Receptor and IL–2 Responsiveness in Human CD4+ T Cells

Fresh human CD8⁺ T cells showed a strong proliferative response to a high concentration of interleukin 2 (IL–2) in the absence of macrophages. In contrast, CD4⁺ T cells revealed no significant IL–2 responsiveness in the absence of macrophages. However, if CD4⁺ T cells were cocultured with macrophages, they showed higher proliferative response to IL–2 than CD8⁺ T cells. In accordance with the magnitude of IL–2 responsiveness, freshly isolated CD8⁺ T cells expressed significant amounts of p75 IL–2 receptor, while fresh CD4⁺ T cells did not express p75 IL–2 receptor. The expression of p75 IL–2 receptor on CD4⁺ T cells was induced by coculture with macrophages. The macrophage–induced p75 IL–2 receptor acquisition was blocked by monoclonal antibody (inAh) against class II antigen. Moreover, the addition of anti–CD4 mAb or anti–class II mAb to the culture caused a great inhibition of IL–2 responsiveness of CD4⁺ T cells. These results strongly suggest that macrophage–T cell interaction through CD4 and/or class II molecules is essential for the expression of p75 IL–2 receptor and IL–2 responsiveness in human CD4⁺, but not CD8⁺ T cells.     

Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis

Hepatic metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a ligand for alpha(v)beta3 integrin and CD44 receptors, is associated with metastasis in several types of cancer. However, the mechanism by which OPN mediates metastasis in colorectal cancer remains unknown. We hypothesized that OPN mediates invasion of colon cancer cells through basement membrane and migration through extracellular matrix (ECM). In this study, we used CT26 murine colon adenocarcinoma cells syngeneic to BALB/c mice to generate cell lines (pS-OPN) in which OPN expression was suppressed through small interfering RNA (siRNA) plasmids. CT26 wild-type cells (WT) and CT26 cells stably expressing murine-mismatch siRNA (pS-MM) served as controls. Western blotting quantified OPN protein levels and our most downregulated clone, pS-OPN-A4, demonstrated a mean 3.0-fold decrease in OPN protein expression versus WT. In vitro cell motility and invasiveness were decreased in pS-OPN-A4 by 3.6-fold (P = 0.004 versus WT) and 4.1-fold (P = 0.01 versus WT), but proliferation was similar amongst cell lines. We demonstrated that OPN suppression significantly correlates with MMP-2 downregulation. In vivo hepatic metastasis was assessed by quantifying liver weights and surface tumor nodules in 33 BALB/c mice (11/group) subjected to intrasplenic injection of tumor cells. pS-OPN-A4 resulted in a 50.4% decrease in mean liver weight compared with WT (3.79 +/- 1.49 g versus 1.88 +/- 1.34 g, P = 0.009). Only 18% of pS-OPN-A4 livers had >20 metastatic surface nodules compared with 89% for WT and 75% for pS-MM-V6. This study demonstrates that RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates CT26 colon cancer metastasis by diminishing tumor cell motility and invasiveness.     

Intermittent chemotherapy can retain the therapeutic potential of anti‐CD137 antibody during the late tumor‐bearing state

Immunomodulating monoclonal antibodies (mAb) can evoke antitumor T‐cell responses, which are attenuated by regulatory T cells (Treg) and myeloid‐derived suppressor cells (MDSC). Treatment with cyclophosphamide (CP) and gemcitabine (GEM) can mitigate the immunosuppression by Treg and MDSC, respectively. In the current study, we examined the antitumor effects of a combination of local injection with anti‐CD137 mAb and intermittent low‐dose chemotherapy using CP and GEM in subcutaneously established CT26 colon carcinoma. Although a significant antitumor effect was observed when local anti‐CD137 mAb therapy (5 μg) was started early in the tumor‐bearing stage (day 10), no therapeutic efficacy was observed when the mAb therapy was started at a later tumor‐bearing stage (day 17). Analyses of the tumor‐infiltrating immune cells revealed that the number of Gr‐1^high/low CD11b⁺ MDSC started to increase 13 days after tumor inoculation, whereas injection with low‐dose (50 mg/kg) CP and GEM mitigated this increase. In addition, although intermittent injections with low‐dose CP and GEM on days 10 and 18 suppressed tumor growth significantly, additional local injections of anti‐CD137 mAb on days 19, 21, and 23 further augmented the therapeutic efficacy. Cytotoxic T lymphocytes reactive to CT26 and a tumor antigen peptide were induced successfully from the spleen cells of tumor‐cured or tumor‐stable mice. In a bilateral tumor inoculation model, this combination therapy achieved systemic therapeutic effects and suppressed the growth of mAb‐untreated tumors. These results suggest that intermittent immunochemotherapy using CP and GEM could retain the therapeutic potential of anti‐CD137 mAb that is normally impaired during the late tumor‐bearing stage.     

Characterization of a subpopulation of colon cancer cells with stem cell‐like properties

The biology of the normal colonic mucosa suggests that colon cancer originates from normal colon stem cells. CD44 cancer stem cells have been identified in breast and prostate cancer, and we therefore examined whether CD44 similarly identified colon cancer stem cells. Initial assays found CD44^hi colon tumor cells to have enhanced soft agar colony‐forming ability. Subsequently, CD44^hi cells isolated from 4 primary colon adenocarcinoma xenografts were found to be highly tumorigenic in immune deficient mice. CD44^hi cells consistently formed tumors with 1,000 cells, and in multiple experiments, as few as 10 and 100 CD44^hi cells formed tumors in 7/10 and 21/28 mice, respectively. In contrast, CD44^? colon tumor cells were either nontumorigenic or 10–50‐fold less tumorigenic. CD44^hi cells could be serially passaged up to 4 times in vivo, suggesting self‐renewal capacity, and formed tumors that recapitulated the heterogeneity of the original patient tumor. CD44^hi cells were significantly enriched for nuclear activated β‐catenin, a key element in normal stem/progenitor cells and in early colon tumor progression. Bromodeoxyuridine (BrdU) labeling studies indicated that CD44^hi cells divide slowly relative to the CD44^? cells, suggesting their tumorigenicity is not simply due to faster proliferation. Aldehyde dehydrogenase (ALDH) sort further increased the tumorigenicity of CD44^hi cells from 2/2 patient tumors, but CD133 tumor cells in our hands did not have increased tumorigenicity. Our observations indicate that CD44 is a marker of stem‐like cells in colon cancer, and support the use of additional markers to further purify colon cancer stem cells. © 2008 Wiley‐Liss, Inc.