Tumor Growth In Immunocompromised Mice Research Articles

Abstract 4291: Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models

Abstract The adhesion G-protein coupled- receptor 1 ADGRB1/Bai1 was identified as a phosphatidylserine receptor that cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. This function of Bai1 was also shown to promote myoblasts fusion. Cell-cell fusion has been demonstrated to occur in normal physiological functions as well as in diseases. Studies have shown in cancer that cell-cell fusion increases tumor heterogeneity, drug resistance, and metastasis as hybrids acquire new properties that allow them to migrate and proliferate at new sites. Activation of ELMO-Dock signaling pathway has been associated with breast cancer metastasis. Moreover, ELMO1 has been identified as a modifier of breast cancer risk for BRCA mutation carriers. Rac1 was also shown to be overexpressed in proliferative breast disease, preinvasive and invasive breast carcinoma as well as lymph node metastases. Our recent studies showed that breast cancer cell fusion with bone marrow-derived mesenchymal stem cells was modulated by hypoxic conditions and promoted by apoptosis. Our hypothesis is that hypoxia stress induced apoptosis activates the phosphatidylserine receptor Bai1, and it signals through the ELMO/DOCK180/Rac to promote breast cancer cell fusion-driven tumor heterogeneity and metastasis. In this study, we determined the ability of Bai1 depleted breast cancer cells to induce tumor growth in immunocompromised mice. We used Bai1 sgRNA to inhibit Bai1 expression in the breast cancer cell MDA-MB-231. These cells (1x 106) together with bone marrow-derived mesenchymal stem cells (MSCs) (1x 106) were injected into the fat pad of the breast of 15 NOD SCID female mice. Control mice (15) received the same cells with no inhibition of Bai1. The mice were monitored for eight weeks. We found that Bai1 inhibition significantly reduced tumor growth in these mice in the course of time (P<0.05). The average tumor weight was also significantly reduced when Bai1 was inhibited (P<0.001). RNA sequencing of tumors generated from these mice revealed a significant increase in the expression of genes driving ferroptosis and a reduction in critical oncogenes in the experiment group compared to the control group. These findings will be helpful in the development of new classes of drugs effective in breast cancer treatment. Grant Support The NIH/NIMHD grant 5U54MD015929-03, the Society for Investigative Dermatology (SID) Freinkel Diversity Fellowship, and NSF Award Notice for Award ID 2142465 (awarded to FNK). Citation Format: Felicite Kamdem Noubissi, Oluwatowin Odubanjo, Hung-Chung Huang, Brenda Ogle, Paul Tchounwou. Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4291.

Ginseng Glucosyl Oleanolate from Ginsenoside Ro, Exhibited Anti-Liver Cancer Activities via MAPKs and Gut Microbiota In Vitro/Vivo.

Ginseng is widely recognized for its diverse health benefits and serves as a functional food ingredient with global popularity. Ginsenosides with a broad range of pharmacological effects are the most crucial active ingredients in ginseng. This study aimed to derive ginseng glucosyl oleanolate (GGO) from ginsenoside Ro through enzymatic conversion and evaluate its impact on liver cancer in vitro and in vivo. GGO exhibited concentration-dependent HepG2 cell death and markedly inhibited cell proliferation via the MAPK signaling pathway. It also attenuated tumor growth in immunocompromised mice undergoing heterograft transplantation. Furthermore, GGO intervention caused a modulation of gut microbiota composition by specific bacterial populations, including Lactobacillus, Bacteroides, Clostridium, Enterococcus, etc., and ameliorated SCFA metabolism and colonic inflammation. These findings offer promising evidence for the potential use of GGO as a natural functional food ingredient in the prevention and treatment of cancer.

The effect of an exopolysaccharide probiotic molecule from Bacillus subtilis on breast cancer cells

IntroductionMany well-known risk factors for breast cancer are associated with dysbiosis (an aberrant microbiome). However, how bacterial products modulate cancer are poorly understood. In this study, we investigated the effect of an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis on breast cancer phenotypes. Although B. subtilis is commonly included in probiotic preparations and its EPS protects against inflammatory diseases, it was virtually unknown whether B. subtilis-derived EPS affects cancer.MethodsThis work investigated effects of EPS on phenotypes of breast cancer cells as a cancer model. The phenotypes included proliferation, mammosphere formation, cell migration, and tumor growth in two immune compromised mouse models. RNA sequencing was performed on RNA from four breast cancer cells treated with PBS or EPS. IKKβ or STAT1 signaling was assessed using pharmacologic or RNAi-mediated knock down approaches. ResultsShort-term treatment with EPS inhibited proliferation of certain breast cancer cells (T47D, MDA-MB-468, HCC1428, MDA-MB-453) while having little effect on others (MCF-7, MDA-MB-231, BT549, ZR-75-30). EPS induced G1/G0 cell cycle arrest of T47D cells while increasing apoptosis of MDA-MB-468 cells. EPS also enhanced aggressive phenotypes in T47D cells including cell migration and cancer stem cell survival. Long-term treatment with EPS (months) led to resistance in vitro and promoted tumor growth in immunocompromised mice. RNA-sequence analysis showed that EPS increased expression of pro-inflammatory pathways including STAT1 and NF-κB. IKKβ and/or STAT1 signaling was necessary for EPS to modulate phenotypes of EPS sensitive breast cancer cells. DiscussionThese results demonstrate a multifaceted role for an EPS molecule secreted by the probiotic bacterium B. subtilis on breast cancer cell phenotypes. These results warrant future studies in immune competent mice and different cancer models to fully understand potential benefits and/or side effects of long-term use of probiotics.

Ketogenic diets slow melanoma growth in vivo regardless of tumor genetics and metabolic plasticity

BackgroundGrowing evidence supports the use of low-carbohydrate/high-fat ketogenic diets as an adjunctive cancer therapy. However, it is unclear which genetic, metabolic, or immunological factors contribute to the beneficial effect of ketogenic diets. Therefore, we investigated the effect of ketogenic diets on the progression and metabolism of genetically and metabolically heterogeneous melanoma xenografts, as well as on the development of melanoma metastases in mice with a functional immune system.MethodsMice bearing BRAF mutant, NRAS mutant, and wild-type melanoma xenografts as well as mice bearing highly metastatic melanoma allografts were fed with a control diet or ketogenic diets, differing in their triglyceride composition, to evaluate the effect of ketogenic diets on tumor growth and metastasis. We performed an in-depth targeted metabolomics analysis in plasma and xenografts to elucidate potential antitumor mechanisms in vivo.ResultsWe show that ketogenic diets effectively reduced tumor growth in immunocompromised mice bearing genetically and metabolically heterogeneous human melanoma xenografts. Furthermore, the ketogenic diets exerted a metastasis-reducing effect in the immunocompetent syngeneic melanoma mouse model. Targeted analysis of plasma and tumor metabolomes revealed that ketogenic diets induced distinct changes in amino acid metabolism. Interestingly, ketogenic diets reduced the levels of alpha-amino adipic acid, a biomarker of cancer, in circulation to levels observed in tumor-free mice. Additionally, alpha-amino adipic acid was reduced in xenografts by ketogenic diets. Moreover, the ketogenic diets increased sphingomyelin levels in plasma and the hydroxylation of sphingomyelins and acylcarnitines in tumors.ConclusionsKetogenic diets induced antitumor effects toward melanoma regardless of the tumors´ genetic background, its metabolic signature, and the host immune status. Moreover, ketogenic diets simultaneously affected multiple metabolic pathways to create an unfavorable environment for melanoma cell proliferation, supporting their potential as a complementary nutritional approach to melanoma therapy.

Abstract LB144: Antagonism of SF-1 as a potential targeted therapy for malignant Leydig cell tumors

Abstract Leydig cell tumors (LCT), originating from androgen-producing interstitial Leydig cells of the testis, represent about 3% of all testicular cancers. LCT belong to the family of sex cord stromal tumors (SCST), a collection of tumors formed in the supporting tissue within the ovaries or testes. Although 90% of LCT are considered benign and can be cured by orchiectomy, in adult patients about 10% of LCT are malignant and metastasis is common. Non-resectable metastatic disease is poorly responsive to radiation and chemotherapy and patients are advised to seek clinical trials. Steroidogenic Factor 1 (SF-1, or NR5A1) is a transcription factor that is essential for the development of the adrenal gland and gonads. SF-1 mutations result in disorders of sexual development, ovarian failure, and adrenal insufficiency. SF-1 is necessary for development of fetal and adult Leydig cells and is strongly expressed in LCT. Significant data support the role of SF-1 in adrenocortical cancer (ACC). To address the need for a targeted therapy in ACC and other SF-1-dependent malignancies, Orphagen has identified potent small molecule antagonists to SF-1*. Here, using R2C, a rat Leydig tumor cell line, we demonstrate that small molecule antagonists of SF-1 inhibit Leydig tumor cell proliferation in vitro and in vivo. OR-449, an orally available inhibitor of SF-1 transcriptional activity (SF-1 Luc IC50 = 16 nM) and OR-907S, a probe SF-1 antagonist (SF-1 Luc IC50 = 22 nM), exhibit striking anti-proliferative activity in R2C cell cultures, inhibiting DNA synthesis by >90% at 1 μM with estimated IC50’s of 0.068 μM and 0.074 μM, respectively. OR-907R, the ~100-fold less active stereoisomer of OR-907S (SF-1 Luc IC50 = 2 μM) was significantly less active in the R2C proliferation assay (estimated IC50 >10 μM). Moreover, in cell lines lacking SF-1 expression, such as HEK293, SF-1 antagonists have no anti-proliferative activity up to 20 μM, suggesting that the anti-proliferative effect on R2C is SF-1-mediated and not due to cytotoxicity. Furthermore, OR-449 completely blocks R2C xenograft tumor growth in immunocompromised mice at an oral dose of 30 mg/kg/day. OR-449 also dose-dependently regulates expression of SF-1 responsive genes, a mRNA signature first identified in R2C culture by comparison of the activity of OR-907S and OR-907R at 1 μM. OR-449 has excellent pharmacokinetic properties and is well-tolerated in repeat dosing toxicity studies in rodents and non-rodents*. These results highlight SF-1 antagonism as a novel targeted therapeutic approach with potential utility in the treatment of LCT and other SCST. OR-449 is currently in IND-enabling studies in order to enter the clinic by the end of 2022. * P. Crowe, et al. A novel steroidogenic factor-1 antagonist, OR-449, as a targeted therapy for adrenocortical cancer. ENDO 2021: J Endocr Soc, Vol5, Supplement_1, A1010 Citation Format: Haiyan Tao, Emily Eastwood, Raymond G. Fox, Neil Raheja, Paul D. Crowe, Scott M. Thacher. Antagonism of SF-1 as a potential targeted therapy for malignant Leydig cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB144.

Extracellular signal regulated kinase 5 and inflammasome in progression of mesothelioma.

Malignant mesothelioma is an aggressive cancer in desperate need of treatment. We have previously shown that extracellular signaling regulated kinase 5 (ERK5) plays an important role in mesothelioma pathogenesis using ERK5 silenced human mesothelioma cells exhibiting significantly reduced tumor growth in immunocompromised mice. Here, we used a specific ERK 5 inhibitor, XMD8-92 in various in vitro and in vivo models to demonstrate that inhibition of ERK5 can slow down mesothelioma tumorigenesis. First, we show a dose dependent toxicity of XMD8-92 to 2 human mesothelioma cell lines growing as a monolayer. We also demonstrate the inhibition of ERK5 phosphorylation in various human mesothelioma cell lines by XMD8-92. We further confirmed the toxicity of XMD8-92 towards mesothelioma cell lines grown as spheroids in a 3-D model as well as in intraperitoneal (immune-competent) and intrapleural (immune-deficient) mouse models with and without chemotherapeutic drugs. To ascertain the mechanism, we explored the role of the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in the process. We found XMD8-92 attenuated naïve and chemotherapeutic-induced inflammasome priming and activation in mesothelioma cells. It can thus be concluded that ERK5 inhibition attenuates mesothelioma tumor growth and this phenomenon in part is regulated by the inflammasome.

Noncalcemic 20-hydroxyvitamin D3 inhibits human melanoma growth in in vitro and in vivo models.

A novel pathway of vitamin D3 (D3) metabolism, initiated by C20-hydroxylation of D3 by CYP11A1, has been confirmed to operate in vivo. Its major product, 20(OH)D3, exhibits antiproliferative activity in vitro comparable to that of 1,25(OH)2D3, but is noncalcemic in mice and rats. To further characterize the antimelanoma activity of 20(OH)D3, we tested its effect on colony formation of human melanoma cells in monolayer culture and anchorage-independent growth in soft agar. The migratory capabilities of the cells and cell-cell and cell-extracellular matrix interactions were also evaluated using transwell cell migration and spheroid toxicity assays. To assess the antimelanoma activity of 20(OH)D3 in vivo, age-matched immunocompromised mice were subcutaneously implanted with luciferase-labelled SKMel-188 cells and were randomly assigned to be treated with either 20(OH)D3 or vehicle (n=10 per group). Tumor size was measured with caliper and live bioimaging methods, and overall health condition expressed as a total body score scale. The following results were observed: (i) 20(OH)D3 inhibited colony formation both in monolayer and soft agar conditions, (ii) 20(OH)D3 inhibited melanoma cells in both transwell migration and spheroid toxicity assays, and (iii) 20(OH)D3 inhibited melanoma tumor growth in immunocompromised mice without visible signs of toxicity. However, although the survival rate was 90% in both groups, the total body score was higher in the treatment group compared to control group (2.8 vs. 2.55). In conclusion, 20(OH)D3, an endogenously produced secosteroid, is an excellent candidate for further preclinical testing as an antimelanoma agent.

Abstract IA02: Molecular pathogenesis of IDH mutant cancers and evolving treatment strategies

Abstract IDH1 and IDH2 mutations have now been described in a variety of cancers including gliomas, acute myelogenous leukemias, chondrosarcomas, cholangiocarcinomas, and angioimmunoblastic T-cell lymphoma. Tumor-associated IDH1 and IDH2 mutants produce large amounts of the R enantiomer of 2-hydroxyglutarate (R-2HG), which accumulates to mM levels in IDH mutant tumor cells. R-2HG modulates (usually inhibits) various 2-oxoglutarate-dependent enzymes, including various 2-oxoglutarate-dependent dioxygenases. Examples of such dioxygenases include the JmjC family of histone demethylases and the TET methylcytosine hydroxylases. The challenge for the field has been to determine which enzymes, when modulated by R-2HG, drive transformation and which are simply bystanders. We developed a cell-based assay for hematopoietic transformation by mutant IDH and showed that transformation in this setting is indeed mediated by R-2HG and that its effects are reversible. Moreover, we and others provided evidence that inhibition of TET2 by R-2HG contributes to leukemic transformation in cells carrying IDH mutations. Small molecules that block R-2HG production appear promising based on early Phase 1 data in IDH1 and IDH2 mutant leukemias. We also developed a model of astrocytic transformation by IDH. We found that activation of the EglN prolyl hydroxylases, which target the HIF transcription factor for destruction, by R-2HG promotes soft agar growth by immortalized astrocytes but is not sufficient to promote tumor growth in immunocompromised mice. More recently we have developed an oncogene cooperation assay in which mutant IDH promotes tumor growth in mice. In addition, we have identified an enzyme in glioma cells that, when inhibited by R-2HG, creates a distinctive metabolic profile and renders IDH mutant cells dependent on glutaminase activity for survival. Citation Format: William G. Kaelin, Jr. Molecular pathogenesis of IDH mutant cancers and evolving treatment strategies. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA02.

Profiling Hsp90 differential expression and the molecular effects of the Hsp90 inhibitor IPI-504 in high-grade glioma models.

Retaspimycin hydrochloride (IPI-504), an Hsp90 (heat shock protein 90) inhibitor, has shown activity in multiple preclinical cancer models, such as lung, breast and ovarian cancers. However, its biological effects in gliomas and normal brain derived cellular populations remain unknown. In this study, we profiled the expression pattern of Hsp90α/β mRNA in stable glioma cell lines, multiple glioma-derived primary cultures and human neural stem/progenitor cells. The effects of IPI-504 on cell proliferation, apoptosis, motility and expression of Hsp90 client proteins were evaluated in glioma cell lines. In vivo activity of IPI-504 was investigated in subcutaneous glioma xenografts. Our results showed Hsp90α and Hsp90β expression levels to be patient-specific, higher in high-grade glioma-derived primary cells than in low-grade glioma-derived primary cells, and strongly correlated with CD133 expression and differentiation status of cells. Hsp90 inhibition by IPI-504 induced apoptosis, blocked migration and invasion, and significantly decreased epidermal growth factor receptor levels, mitogen-activated protein kinase and/or Akt activities, and secretion of vascular endothelial growth factor in glioma cell lines. In vivo study showed that IPI-504 could mildly attenuate tumor growth in immunocompromised mice. These findings suggest that targeting Hsp90 by IPI-504 has the potential to become an active therapeutic strategy in gliomas in a selective group of patients, but further research into combination therapies is still needed.

Protumorigenic actions of S100A2 involve regulation of PI3/Akt signaling and functional interaction with Smad3

S100 family of calcium-binding proteins is commonly upregulated in a variety of tumor types and is often associated with tumor progression. Among several S100 members, altered expression of S100A2 is a potential diagnostic and prognostic marker in cancer. Several reports suggest a role for S100A2 in metastasis. Earlier, our studies established regulation of S100A2 by transforming growth factor-β (TGF-β) and its involvement in TGF-β-mediated cancer cell invasion and migration. However, the molecular mechanisms of S100A2 protumorigenic actions remain unexplored. In the present study, we demonstrate that overexpression of S100A2 in A549 lung cancer cells induced epithelial-mesenchymal transition (EMT) followed by increased invasion, loose colony morphology in soft agar and enhanced Akt phosphorylation (Ser-473). Furthermore, overexpression of S100A2 led to increased tumor growth in immunocompromised mice. In agreement, immunohistochemical examination of resected xenograft tumors established inverse correlation between S100A2 and E-cadherin expression together with activated Akt signaling. Interestingly, our study demonstrates a strong dependence of S100A2 and Smad3 in TGF-β-induced Hep3B cell EMT and invasion. Most importantly, we demonstrate that these effects of S100A2 are manifested through functional interaction with Smad3, which is enhanced in the presence of high calcium and TGF-β. S100A2 stabilizes Smad3 and binds to its C-terminal MH2 domain. Additionally, loss of S100A2 attenuates the transcription of TGF-β/Smad3 target genes involved in tumor promotion, such as PA1-1 and vimentin. Collectively, our findings present the first mechanistic details of S100A2 protumorigenic actions and its involvement in TGF-β-mediated cancer cell invasion and EMT.

TRIM11 is overexpressed in high-grade gliomas and promotes proliferation, invasion, migration and glial tumor growth

TRIM11 (tripartite motif-containing protein 11), an E3 ubiquitin ligase, is known to be involved in the development of the central nervous system. However, very little is known regarding the role of TRIM11 in cancer biology. Here, we examined the expression profile of TRIM11, along with two stem cell markers CD133 and nestin, in multiple glioma patient specimens, glioma primary cultures derived from tumors taken at surgery and normal neural stem/progenitor cells (NSCs). The oncogenic function of TRIM11 in glioma biology was investigated by knockdown and/or overexpression in vitro and in vivo experiments. Our results showed that TRIM11 expression levels were upregulated in malignant glioma specimens and in high-grade glioma-derived primary cultures, whereas remaining low in glioblastoma multiforme (GBM) stable cell lines, low-grade glioma-derived primary cultures and NSCs. The expression pattern of TRIM11 strongly correlated with that of CD133 and nestin and differentiation status of malignant glioma cells. Knock down of TRIM11 inhibited proliferation, migration and invasion of GBM cells, significantly decreased epidermal growth factor receptor (EGFR) levels and mitogen-activated protein kinase activity, and downregulated HB-EGF (heparin-binding EGF-like growth factor) mRNA levels. Meanwhile, TRIM11 overexpression promoted a stem-like phenotype in vitro (tumorsphere formation) and enhanced glial tumor growth in immunocompromised mice. These findings suggest that TRIM11 might be an indicator of glioma malignancy and has an oncogenic function mediated through the EGFR signaling pathway. TRIM11 overexpression potentially leads to a more aggressive glioma phenotype, along with increased malignant tumor growth and poor survival. Taken together, clarification of the biological function of TRIM11 and pathways it affects may provide novel therapeutic strategies for treating malignant glioma patients.

A Combined DNA-Affinic Molecule and N-Mustard Alkylating Agent Has an Anti-Cancer Effect and Induces Autophagy in Oral Cancer Cells

Although surgery or the combination of chemotherapy and radiation are reported to improve the quality of life and reduce symptoms in patients with oral cancer, the prognosis of oral cancer remains generally poor. DNA alkylating agents, such as N-mustard, play an important role in cancer drug development. BO-1051 is a new 9-anilinoacridine N-mustard-derivative anti-cancer drug that can effectively target a variety of cancer cell lines and inhibit tumorigenesis in vivo. However, the underlying mechanism of BO-1051-mediated tumor suppression remains undetermined. In the present study, BO-1051 suppressed cell viability with a low IC50 in oral cancer cells, but not in normal gingival fibroblasts. Cell cycle analysis revealed that the tumor suppression by BO-1051 was accompanied by cell cycle arrest and downregulation of stemness genes. The enhanced conversion of LC3-I to LC3-II and the formation of acidic vesicular organelles indicated that BO-1501 induced autophagy. The expression of checkpoint kinases was upregulated as demonstrated with Western blot analysis, showing that BO-1051 could induce DNA damage and participate in DNA repair mechanisms. Furthermore, BO-1051 treatment alone exhibited a moderate tumor suppressive effect against xenograft tumor growth in immunocompromised mice. Importantly, the combination of BO-1051 and radiation led to a potent inhibition on xenograft tumorigenesis. Collectively, our findings demonstrated that BO-1051 exhibited a cytotoxic effect via cell cycle arrest and the induction of autophagy. Thus, the combination of BO-1051 and radiotherapy may be a feasible therapeutic strategy against oral cancer in the future.

Sustained inhibition of tumor growth and prolonged survival following sequential administration of doxorubicin and zoledronic acid in a breast cancer model

Combination therapy, using agents that target the microenvironment as well as the cancer cells, is common in the treatment of advanced breast cancer. Here, we show that a 6-week course of weekly sequential administration of the cytotoxic drug doxorubicin (2 mg/kg), followed 24 hr later by the antiresorptive agent zoledronic acid (100 microg/kg), causes substantial inhibition of subcutaneous MDA-MB-436 breast tumor growth in immunocompromised mice, leading to significantly increased survival. Tumor growth did not resume following withdrawal of treatment after 6 weeks, with 60% of the animals in this group surviving for more than 160 days. In comparison, animals receiving single-agent therapy all died within 50 days. Molecular analysis of the tumors showed no effect on cell cycle or apoptosis following administration of 100 microg/kg zoledronic acid or 2 mg/kg doxorubicin alone. When doxorubicin was administered 24 hr before zoledronic acid, tumors displayed decreased expression of CYCLINS E1, B, D1 and D3 as well as CDK2, CDC2, CDK4 and CDK7, indicative of cell-cycle inhibition. Tumors from animals receiving sequential treatment also showed induction of both intrinsic- and extrinsic-apoptotic pathways, with increased expression of BAX, decreased expression of BCL-2 and activation of CASPASE 3, 8 and 9. Accumulation of the unprenylated form of RAP1a, a surrogate marker for uptake of zoledronic acid, was only detected in tumors from animals treated with doxorubicin 24 hr before zoledronic acid. Our data are the first to show a sustained antitumor effect in vivo following a limited course of sequential administration of doxorubicin followed by zoledronic acid.

Cancer stem cells in solid tumors

Cancer stem cells (CSCs) are cells that drive tumorigenesis, as well as giving rise to a large population of differentiated progeny that make up the bulk of the tumor, but that lack tumorigenic potential. CSCs have been identified in a variety of human tumors, as assayed by their ability to initiate tumor growth in immunocompromised mice. Further characterization studies have demonstrated that gene expression profiles in breast cancer correlate with patient prognosis, and brain CSCs are specifically resistant to radiation through DNA damage repair. In addition, specific signaling pathways play a functional role in CSC self renewal and/or differentiation, and early studies indicate that CSCs are associated with a microenvironmental niche. Thus the biological properties of CSCs are just beginning to be revealed, and the continuation of these studies should lead to the development of CSC-targeted therapies for cancer treatment.

Bioregulators Come of Age in the Control of Tumor Growth and Metastasis

During the last few years, there has been a resurgence of interest in the development of new approaches for the biological control of tumor growth and metastasis. This situation is due, in no small part, to our increased understanding of the roles of various growth factors and inhibitors, cytokines, interferons, and other bioregulators in controlling tumor cell proliferation and other properties important in cancer. Systemic administration of bioregulators has resulted in significant inhibition of tumor growth and, in some cases, in complete regression of tumors (7). Unfortunately, many of the most promising bioregulators, such as the interferons, possess a variety of in vivo side effects that can limit their clinical usefulness. Moreover, their pharmacokinetics are often undesirable, and other approaches, such as stimulating macrophage cytolytic function, once touted to be a panacea for controlling tumor growth and metastasis, have been generally unsuccessful in clinical settings. In a report published in this issue of the Journal, Kundu et al. (2) show that interleukin 10 (IL-10), an acid-sensitive protein of 35-40 kd originally described as an immunosuppressive cytokine, might be a useful new weapon for suppressing tumor growth and metastasis. This finding is a pleasant surprise. IL-10 has pleiotropic effects and is capable of inhibiting macrophage function and T-cell cytokine production, resulting in suppression of helper T-cell subpopulations, delayed-type hypersensitivity reactions, and other mechanisms of cellular immunity. From these properties, one would not expect much antineoplastic activity from IL-10. On the other hand, IL-10 can stimulate certain B-cell subpopulations as well as interleukin 2-regulated T-cell subpopulations, such as natural killer (NK) cells (3). These latter properties may be more important in inhibiting tumor growth and metastatic spread. That IL-10 can suppress tumor growth and macrophage infiltration in vivo was shown by Richter et al. (4) using IL-10 gene-transfected hamster CHO cells implanted in nude mice. Although these cells are not directly inhibited by IL-10, the transfected cells apparently produce and secrete enough IL-10 to cause host-mediated inhibition of tumor growth and inhibition of macrophage infiltration into tumors in a xenograft model. Similarly, Allione et al. (5) transfected various cytokine genes, including IL-10, into a murine mammary adenocarcinoma cell line and examined the growth properties of the transfected cells in syngeneic mice. They also found that IL-10 gene transfection resulted in inhibition of tumor growth. Against this background and with the observation that IL-10 administration is not associated with the systemic toxicity often found with other cytokines (6), Kundu et al. (2) evaluated the ability of IL-10 to inhibit murine mammary tumor growth and metastasis in syngeneic immunocompetent and immunocompromised mice. In their study, two transplantable, highly malignant mammary tumor cell lines that are capable of forming experimental and spontaneous metastases in mice were transfected to express and produce IL-10; one cell line was highly immunogenic, and the other was poorly immunogenic in syngeneic animals. The growth of neither of the cell lines was directly affected by IL-10. Tumor growth and metastasis were evaluated by inoculating syngeneic mice or SCID or SCID/Beige immunocompromised mice subcutaneously or intravascularly with IL-10or neo-transfected tumor cells (18 different cell clones that contained the IL-10 gene). As was found previously with other tumor cell types (4,5), the growth of the IL-10 gene-transfected highly immunogenic mammary tumor cells was completely inhibited in immunocompetent, syngeneic animals and was significantly inhibited in the immunocompromised mice. Moreover, although there was some tumor growth in immunocompromised mice, formation of experimental and spontaneous metastasis was completely inhibited, suggesting a different mechanism for inhibition of metastasis. In support of this hypothesis, Kundu et al. (2) suppressed NK activity by injecting anti-asialo-GMl ganglioside antibody into syngeneic BALB/cBy mice and found that the antimetastatic activity of IL-10 was reversed. Furthermore, IL-10 could not control metastasis in SCID/Beige mice that also lack NK function. NK activity is known to be important in suppressing bloodborne metastasis, and Kundu et al. (2) did not find any differences in the adhesion and invasion properties of the IL-10 gene-transfected cells; therefore, the suppression of metastasis formation was likely due, at least in part, to IL-10-stimulated

Antimetastatic and antitumor activities of interleukin 10 in a murine model of breast cancer.

Interleukin 10 (IL-10) is a potent immunoregulatory cytokine. It inhibits some cell functions, including T-helper (Th1) cell activity (i.e., interleukin 2 and interferon gamma production), and stimulates other functions such as a natural killer (NK) activity. In mice, IL-10 suppresses tumorigenicity in a xenograft system using a nonmetastasizing hamster cell line. We evaluated the antitumor and antimetastatic properties of IL-10 in syngeneic immunocompetent and immunocompromised murine hosts. Using the plasmids pBMGneo and pBMGneo.IL-10, we transfected the highly malignant murine mammary tumor cell lines 410.4 and 66.1 (transfectants designated as 410.4-IL10 and 66.1-IL10, respectively) to stably express IL-10 (2-100 U IL-10/2.5 x 10(5) cells per 48 hours). Tumorigenic and metastatic activities of the parent and transfected cells w ere measured in immunocompetent, syngeneic BALB/cByJ mice as well as in immunocompromised C.B-17/IcrCrl-SCID/Beige mice. Tumor growth was completely inhibited following inoculations of 5 x 10(6)410.4-IL10 cells in immunocompetent, syngeneic BALB/cByJ mice. This inoculum contains 100 times the minimum cell number required for 100% tumor incidence. In contrast, tumor growth following the inoculation of parental 410.4 or 410.4-neo cells was progressive, resulting in death of animals from pulmonary metastases at days 40-50 and transplantation. The tumorigenicity of 66.1-IL-10, compared with that of its parent cell line, was also significantly abrogated by IL-10 expression. Furthermore, in immunocompetent mice, the metastatic potential of both 410.4-IL10 and 66.1-IL10 was also completely inhibited. In immunocompromised C.B-17/IcrCrl-SCID/BR or C.B-17/IcrCrl-SCID/Beige mice, subcutaneous implants of 410.4-IL10 grew progressively, but growth was inhibited significantly in comparison to that produced by the parental 410.4 or 410.4-neo cells. In spite of the more limited efficacy of IL-10 against tumor growth in immunocompromised mice, spontaneous metastasis of 410.4-IL10 cells in C.B-17/IcrCrl-SCID/BR mice was inhibited by 90%. When NK activity was suppressed by asialoGM1 ganglioside antibody in BALB/cByJ mice or in C.B-17/IcrCrl-SCID/Beige mice, the antimetastatic effect of IL-10 was lost. These data show for the first time that IL-10 is a potent antimetastatic agent that is effective in immunocompromised hosts. This effect thus appears to be relatively independent of T-cell function but is dependent on NK activity. In contrast, the inhibitory effect of IL-10 on tumorigenicity relies on T-cell function. Based on the recent observation of others that IL-10 has little toxicity when administered systemically to human volunteers and also on the findings of this study that it has antitumor and antimetastitic properties in mice, possible use of IL-10 in the treatment of human metastatic cancers deserves consideration.

The immunogenic properties of drug-resistant murine tumor cells do not correlate with expression of the MDR phenotype.

Alterations in the immunogenic properties of tumor cells frequently accompany selection for multiple-drug-resistant (MDR) variants. Therefore, studies were performed to examine the hypothesis that overexpression of membrane P-glycoprotein, commonly observed in MDR tumor cells, is associated with enhanced immunogenic properties. Immunogenicity was determined by (a) the ability of drug-sensitive parental UV2237M fibrosarcoma cells and drug-resistant UV2237M variant cells to immunize normal mice against rechallenge with parental tumor cells and (b) the ability of normal syngeneic mice to reject cell inocula that caused progressive tumor growth in immunocompromised mice. Variant UV2237M cell lines included subpopulations selected for a six- to ten-fold increase in mRNA for P-glycoprotein and expression of the MDR phenotype (resistance to doxorubicin) and cells sensitive to doxorubicin (and no expression of MDR properties) but resistant to ouabain. All UV2237M drug-resistant cells were highly immunogenic in immunocompetent mice, regardless of their MDR phenotype. Additional studies showed that CT-26 murine adenocarcinoma cells, sensitive or resistant to doxorubicin (expressing high levels of P-glycoprotein), injected into normal syngeneic Balb/c mice produced rapidly growing tumors. The data do not demonstrate a correlation between the immunogenic properties of drug-resistant tumor cells and the expression of P-glycoprotein.