Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.

Discovery of 1-(1H-Pyrazolo[4,3-c]pyridin-6-yl)urea Inhibitors of Extracellular Signal-Regulated Kinase (ERK) for the Treatment of Cancers.

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF(V600E) xenograft model.

Spontaneous reversion of the angiogenic phenotype to a nonangiogenic and dormant state in human tumors.

UNLABELLED: The angiogenic switch, a rate-limiting step in tumor progression, has already occurred by the time most human tumors are detectable. However, despite significant study of the mechanisms controlling this switch, the kinetics and reversibility of the process have not been explored. The stability of the angiogenic phenotype was examined using an established human liposarcoma xenograft model. Nonangiogenic cells inoculated into immunocompromised mice formed microscopic tumors that remained dormant for approximately 125 days (vs. <40 days for angiogenic cells) whereupon the vast majority (>95%) initiated angiogenic growth with second-order kinetics. These original, clonally derived angiogenic tumor cells were passaged through four in vivo cycles. At each cycle, a new set of single-cell clones was established from the most angiogenic clone and characterized for in vivo for tumorigenic activity. A total of 132 single-cell clones were tested in the second, third, and fourth in vivo passage. Strikingly, at each passage, a portion of the single-cell clones formed microscopic, dormant tumors. Following dormancy, like the original cell line, these revertant tumors spontaneously switched to the angiogenic phenotype. Finally, revertant clones were transcriptionally profiled and their angiogenic output determined. Collectively, these data demonstrate that the angiogenic phenotype in tumors is malleable and can spontaneously revert to the nonangiogenic phenotype in a population of human tumor cells. IMPLICATIONS: Leveraging the rate of reversion to the nonangiogenic phenotype and tumor dormancy may be a novel anticancer strategy.

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer.

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.

Combined vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance.

PURPOSE: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non-small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. EXPERIMENTAL DESIGN: We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. RESULTS: Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. CONCLUSION: These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

Tumor dormancy due to failure of angiogenesis: role of the microenvironment.

Tumor progression is dependent on a number of sequential steps, including initial recruitment of blood vessels (i.e., angiogenic switch). Failure of a microscopic tumor to complete one or more of these early steps may lead to delayed clinical manifestation of the cancer. In this review we summarize some of the clinical and experimental evidence suggesting that microscopic human cancers can remain in an asymptomatic, non-detectable, and occult state for the life of a person or animal. We present three clinical cases where tumors present shortly after an accidental trauma in otherwise healthy individuals. We also review current experimental human tumor dormancy models with special emphasis on the angiogenic switch which closely recapitulates clinically observed delay in tumor recurrence.

Tumor necrosis is associated with increased alphavbeta3 integrin expression and poor prognosis in nodular cutaneous melanomas.

BACKGROUND: Tumor necrosis and apoptotic activity are considered important in cancer progression, but these features have not been much studied in melanomas. Our hypothesis was that rapid growth in cutaneous melanomas of the vertical growth phase might lead to tissue hypoxia, alterations in apoptotic activity and tumor necrosis. We proposed that these tumor characteristics might be associated with changes in expression of cell adhesion proteins leading to increased invasive capacity and reduced patient survival. METHODS: A well characterized series of nodular melanoma (originally 202 cases) and other benign and malignant melanocytic tumors (109 cases) were examined for the presence of necrosis, apoptotic activity (TUNEL assay), immunohistochemical expression of hypoxia markers (HIF-1 alpha, CAIX, TNF-alpha, Apaf-1) and cell adhesion proteins (alphavbeta3 integrin, CD44/HCAM and osteopontin). We hypothesized that tumor hypoxia and necrosis might be associated with increased invasiveness in melanoma through alterations of tumor cell adhesion proteins. RESULTS: Necrosis was present in 29% of nodular melanomas and was associated with increased tumor thickness, tumor ulceration, vascular invasion, higher tumor proliferation and apoptotic index, increased expression of alphavbeta3 integrin and poor patient outcome by multivariate analysis. Tumor cell apoptosis did also correlate with reduced patient survival. Expression of TNF-alpha and Apaf-1 was significantly associated with tumor thickness, and osteopontin expression correlated with increased tumor cell proliferation (Ki-67). CONCLUSION: Tumor necrosis and apoptotic activity are important features of melanoma progression and prognosis, at least partly through alterations in cell adhesion molecules such as increased alphavbeta3 integrin expression, revealing potentially important targets for new therapeutic approaches to be further explored.

Tumor-vascular interactions and tumor dormancy.

Tumor progression is dependent on a number of sequential steps, including initial tumor-vascular interactions and recruitment of blood vessels (i.e., the angiogenic switch), as well as tumor cells interacting with the surrounding microenvironment and its different components. Failure of a microscopic tumor to complete one or more of these early stages may lead to delayed clinical manifestation of the cancer and a state of stable non-progressing disease (i.e., tumor dormancy). In this review, some of the clinical and experimental evidence is summarized, suggesting that microscopic human cancers, either primary, recurrent or metastatic, can remain in an asymptomatic, non-detectable, and occult state for a long period of time. We also review current experimental human tumor dormancy models which closely recapitulate clinically observed delay in tumor progress.

Platelet-associated PF-4 as a biomarker of early tumor growth.

Early tumor detection and intervention are important determinants of survival in patients with cancer. We have recently reported that the "platelet angiogenesis proteome" may be used to detect microscopic tumors in mice. We now present evidence that changes in platelet-associated platelet factor-4 (PF-4) detect malignant growth across a spectrum of human cancers in mice. A deregulated expression of an 8206-Da protein was observed by surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-ToF MS) proteomic comparison of platelets from normal and tumor-bearing mice. The differentially expressed protein was identified as PF-4 by tandem mass spectrometry and ProteinChip immunoassay using anti-PF-4 antibody. The platelet-associated PF-4 appeared to be up-regulated in early growth of human liposarcoma, mammary adenocarcinoma, and osteosarcoma. A 120-day follow-up study of liposarcoma revealed a sustained 2-fold or higher increase of platelet-associated PF-4 at 19, 30, and 120 days. In contrast, only an insignificant change of PF-4 was observed in the plasma of mice bearing the different human tumor xenografts, and throughout the 120 days of the liposarcoma study. We conclude that platelet-associated PF-4, but not its plasma counterpart, may represent a potential biomarker of early tumor presence.

PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members.

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer.

EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.

Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch.

Tumor progression depends on sequential events, including a switch to the angiogenic phenotype (i.e., initial recruitment of blood vessels). Failure of a microscopic tumor to complete one or more early steps in this process may lead to delayed clinical manifestation of the cancer. Microscopic human cancers can remain in an asymptomatic, non-detectable, and occult state for the life of a person. Clinical and experimental evidence suggest that human tumors can persist for long periods of time as microscopic lesions that are in a state of dormancy (i.e., not expanding in tumor mass). Because it is well established that tumor growth beyond the size of 1-2 mm is angiogenesis-dependent, we hypothesized that presentation of large tumors is attributed to a switch to the angiogenic phenotype in otherwise microscopic, dormant tumors. Although clinically important, the biology of human tumor dormancy is poorly understood. The development of animal models which recapitulate the clinically observed timing and proportion of dormant tumors which switch to the angiogenic phenotype are reviewed here. The contributing molecular mechanisms involved in the angiogenic switch and different strategies for isolation of both angiogenic and non-angiogenic tumor cell populations from otherwise heterogeneous human tumor cell lines or surgical specimens are also summarized. Several imaging techniques have been utilized for the qualitative and quantitative detection of microscopic tumors in mice and their strengths and limitations are discussed. The animal models employed here permitted further studies of the angiogenic switch. These models also allowed development of an angiogenesis-based panel of blood and urine biomarkers that can be quantified and used to detect microscopic tumors before or during the angiogenic switch. If the information obtained from these animal models is translatable to the clinic, it may be possible in the future to liberate the management of cancer from a dependency on anatomical site years before it becomes symptomatic and detectable.

Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis.

The disease state of cancer appears late in tumor development. Before being diagnosed, a tumor can remain for prolonged periods of time in a dormant state. Dormant human cancer is commonly defined as a microscopic tumor that does not expand in size and remains asymptomatic. Dormant tumors represent an early stage in tumor development and may therefore be a potential target for nontoxic, antiangiogenic therapy that could prevent tumor recurrence. Here, we characterize an experimental model that recapitulates the clinical dormancy of human tumors in mice. We demonstrate that these microscopic dormant cancers switch to the angiogenic phenotype at a predictable time. We further show that while angiogenic liposarcomas expand rapidly after inoculation of tumor cells in mice, nonangiogenic dormant liposarcomas remain microscopic up to one-third of the normal severe combined immune deficiency (SCID) mouse life span, although they contain proliferating tumor cells. Nonangiogenic dormant tumors follow a similar growth pattern in subcutaneous (s.c.) and orthotopic environments. Throughout the dormancy period, development of intratumoral vessels is impaired. In nonangogenic dormant tumors, small clusters of endothelial cells without lumens are observed early after tumor cell inoculation, but the nonangiogenic tumor cannot sustain these vessels, and they disappear within weeks. There is a concomitant decrease in microvessel density, and the nonangiogenic dormant tumor remains harmless to the host. In contrast, microvessel density in tumors increases rapidly after the angiogenic switch and correlates with rapid expansion of tumor mass. Both tumor types cultured in vitro contain fully transformed cells, but only cells from the nonangiogenic human liposarcoma secrete relatively high levels of the angiogenesis inhibitors thrombospondin-1 and TIMP-1. This model suggests that as improved blood or urine molecular biomarkers are developed, the microscopic, nonangiogenic, dormant phase of human cancer may be vulnerable to antiangiogenic therapy years before symptoms, or before anatomical location of a tumor can be detected, by conventional methods.

A model of human tumor dormancy: an angiogenic switch from the nonangiogenic phenotype.

BACKGROUND: Microscopic human cancers can remain dormant for life. Tumor progression depends on sequential events, including a switch to the angiogenic phenotype, i.e., initial recruitment of new vessels. We previously demonstrated that human tumors contain tumor cell populations that are heterogeneous in angiogenic activity. Here, we separated angiogenic from nonangiogenic human tumor cell populations and compared their growth. METHODS: Severe combined immunodeficient (SCID) mice were inoculated with nonangiogenic human MDA-MB-436 breast adenocarcinoma, KHOS-24OS osteosarcoma, or T98G glioblastoma cells. Most of the resulting tumors remained microscopic (<1 mm diameter), but some eventually became angiogenic and enlarged and were used to isolate angiogenic tumor cells. Angiogenic and nonangiogenic tumor cells were inoculated into SCID mice, and time to the development of palpable tumors was determined. Cell proliferation was assayed in vitro by growth curves and in vivo by staining for proliferating cell nuclear antigen or Ki67. Microscopic tumors from both tumor cell populations were examined for histologic evidence of vascular development 14 days after inoculation in mice. Expression of the angiogenesis inhibitor thrombospondin-1 was examined by immunoblotting. RESULTS: Nonangiogenic tumors of each tumor type developed palpable tumors after means of 119 days (range: 53-185 days) for breast cancer, 238 days (184-291 days) for osteosarcoma, and 226 days (150-301 days) for glioblastoma. Angiogenic cells developed palpable tumors within 20 days after inoculation. However, nonangiogenic and angiogenic cells of each tumor type had similar proliferation rates. Fourteen days after tumor cell inoculation, tumors from angiogenic cells showed evidence of functional vasculature. In contrast, nonangiogenic tumors remained microscopic in size with absent or nonfunctional vasculature. Thrombospondin-1 expression was statistically significantly lower (by five- to 23-fold, depending on tumor type) in angiogenic than nonangiogenic cells. CONCLUSIONS: This model provides a conceptual framework and a reproducible in vivo system to study unresolved central questions in cancer biology regarding the initiation, reversibility, and molecular regulation of the timing of the angiogenic switch.

Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: implications for use as a surrogate marker of antiangiogenic activity.

PURPOSE: Circulating endothelial cells (CEC) comprise at least two distinct populations: bone marrow-derived circulating endothelial progenitors (CEP) and mature CECs derived from existing vasculature. We hypothesized that antiangiogenic agents may have differential effects on CEPs and mature CECs and that these changes may serve as a marker of biological activity. EXPERIMENTAL DESIGN: The effect of angiogenesis inhibitors on CECs was evaluated by flow cytometry after vascular endothelial growth factor (VEGF)-induced mobilization and in mice bearing Lewis lung carcinoma (LLC). Tumor angiogenesis was evaluated in parallel by immunohistochemistry. RESULTS: In nontumor-bearing mice, VEGF administration increased both mature CECs and CEPs. This increase was inhibited by the VEGF receptor 2 inhibitor ZD6474 as well as the VEGF inhibitor-soluble Flt-1. ZD6474 had no significant effect on CECs in the absence of exogenous VEGF stimulation. In contrast, LLC-bearing mice had an increase in mature CECs but not CEPs after 3 days of treatment with ZD6474. The increase in mature CECs was dose-dependent, accompanied by a decrease in tumor microvessel density, and preceded reduction in tumor volume. Treatment of LLC-bearing mice with the vascular targeting agent ZD6126 also increased mature CECs. CONCLUSIONS: VEGF inhibitors can have differential effects on mature CECs and CEPs, and agents inhibiting tumor angiogenesis may cause a concomitant increase in mature CECs. This increase occurs in tumor-bearing but not in nontumor-bearing mice, suggesting that tumor endothelium is a potential source of mature CECs. Therefore, assessing both mature CECs and CEPs may provide insights into the mechanism of antiangiogenic agents and serve as an early surrogate marker of biological activity.

The role of apoptosis in tumor progression and metastasis.

Metastasis, the process by which cancer spreads from a primary to a secondary site, is responsible for the majority of cancer related deaths. Yet despite the detrimental effects of metastasis, it is an extremely inefficient process by which very few of the cells that leave the primary tumor give rise to secondary tumors. Metastasis can be considered as a series of sequential steps that begins with a cell leaving a primary tumor, and concludes with the formation of a metastatic tumor in a distant site. During the process of metastasis cells are subjected to various apoptotic stimuli. Thus, in addition to genetic changes that promote unregulated proliferation, successful metastatic cells must have a decreased sensitivity to apoptotic stimuli. As many cancer cells exhibit aberrations in the level and function of key apoptotic regulators, exploiting these alterations to induce tumor cell apoptosis offers a promising therapeutic target. This review will examine the apoptotic regulators that are often aberrantly expressed in metastatic cells; the role that these regulators may play in metastasis; the steps of metastasis and their susceptibility to apoptosis; and finally, current and future cancer prognostics and treatment targets based on apoptotic regulators.

Ineffectiveness of doxorubicin treatment on solitary dormant mammary carcinoma cells or late-developing metastases.

Breast cancer is noted for long periods of tumor dormancy and metastases can occur many years after treatment. Adjuvant chemotherapy is used to prevent metastatic recurrence but is not always successful. As a model for studying mechanisms of dormancy, we have used two murine mammary carcinoma cell lines: D2.0R/R cells, which are poorly metastatic but form metastases in some mice after long latency times, and D2A1/R cells, which form more numerous metastases much earlier. Previously we identified a surprisingly large population of dormant but viable solitary cells, which persisted in an undivided state for up to 11 weeks after injection of D2.0R/R cells. Dormant cells were also detected for D2A1/R cells, in a background of growing metastases. Here we used this model to test the hypothesis that dormant tumor cells would not be killed by cytotoxic chemotherapy that targets actively dividing cells, and that the late development of metastases from D2.0R/R cells would not be inhibited by chemotherapy that effectively inhibited D2A1/R metastases. We injected mice with D2A1/R or D2.0R/R cells via a mesenteric vein to target liver. We developed a doxorubicin (DXR) treatment protocol that effectively reduced the metastatic tumor burden from D2A1/R cells at 3 weeks. However, this treatment did not reduce the numbers of solitary dormant cells in mice injected with either D2A1/R or D2.0R/R cells. Furthermore, DXR did not reduce the metastatic tumor burden after an 11-week latency period in mice injected with D2.0R/R cells. Thus, apparently effective chemotherapy may spare non-dividing cancer cells, and these cells may give rise to metastases at a later date. This study has important clinical implications for patients being treated with cytotoxic chemotherapy.