In Vitro to In Vivo Concordance of Toxicity Using the Human Proximal Tubule Cell Line HK-2.

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo, we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

Genetically Engineered Human Kidney Cells for Real-Time Cytotoxicity Testing In Vitro.

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.

Diglycolic acid induces HepG2/C3A liver cell toxicity in vitro.

Carboxymethyl starches are added to food products for thickening or tablet binding/filling purposes. Although they lack toxicity, their synthesis creates the chemical byproduct diglycolic acid (DGA), which is difficult to eliminate and whose toxicity is in question. A rare case of an accidental direct exposure to extremely high concentrations of DGA in a person revealed that DGA has the potential to be toxic to several organs, with the kidneys and liver being the most affected organs. Given that DGA is present in our food supply as a chemical byproduct of carboxymethyl starch food additives, we sought to perform in vitro testing of its potential hepatotoxicity to help complement a recent in vivo rat acute dose-response study that also tested for the potential hepatotoxic effects of daily DGA ingestion by oral gavage over a period of 28 days. Using the HepG2/C3A cellular in vitro model, we tested how escalating doses of DGA exposure over 24 h could induce hepatotoxicity. Both in vitro and in vivo testing systems revealed that DGA is indeed a hepatotoxin once a certain exposure threshold is reached. The concordance of these models highlights the utility of in vitro testing to support and help predict in vivo findings.

High-Dose Sirolimus and Immune-Selective Pentostatin plus Cyclophosphamide Conditioning Yields Stable Mixed Chimerism and Insufficient Graft-versus-Tumor Responses.

PURPOSE: We hypothesized that lymphoid-selective host conditioning and subsequent adoptive transfer of sirolimus-resistant allogeneic T cells (T-Rapa), when combined with high-dose sirolimus drug therapy in vivo, would safely achieve antitumor effects while avoiding GVHD. EXPERIMENTAL DESIGN: Patients (n = 10) with metastatic renal cell carcinoma (RCC) were accrued because this disease is relatively refractory to high-dose conditioning yet may respond to high-dose sirolimus. A 21-day outpatient regimen of weekly pentostatin (P; 4 mg/m(2)/dose) combined with daily, dose-adjusted cyclophosphamide (C; ≤200 mg/d) was designed to deplete and suppress host T cells. After PC conditioning, patients received matched sibling, T-cell-replete peripheral blood stem cell allografts, and high-dose sirolimus (serum trough target, 20-30 ng/mL). To augment graft-versus-tumor (GVT) effects, multiple T-Rapa donor lymphocyte infusions (DLI) were administered (days 0, 14, and 45 posttransplant), and sirolimus was discontinued early (day 60 posttransplant). RESULTS: PC conditioning depleted host T cells without neutropenia or infection and facilitated donor engraftment (10 of 10 cases). High-dose sirolimus therapy inhibited multiple T-Rapa DLI, as evidenced by stable mixed donor/host chimerism. No antitumor responses were detected by RECIST criteria and no significant classical acute GVHD was observed. CONCLUSIONS: Immune-selective PC conditioning represents a new approach to safely achieve alloengraftment without neutropenia. However, allogeneic T cells generated ex vivo in sirolimus are not resistant to the tolerance-inducing effects of in vivo sirolimus drug therapy, thereby cautioning against use of this intervention in patients with refractory cancer.

Human kidney proximal tubule cells are vulnerable to the effects of Rauwolfia serpentina.

Rauwolfia serpentina (or Snake root plant) is a botanical dietary supplement marketed in the USA for maintaining blood pressure. Very few studies have addressed the safety of this herb, despite its wide availability to consumers. Its reported pleiotropic effects underscore the necessity for evaluating its safety. We used a human kidney cell line to investigate the possible negative effects of R. serpentina on the renal system in vitro, with a specific focus on the renal proximal tubules. We evaluated cellular and mitochondrial toxicity, along with a variety of other kidney-specific toxicology biomarkers. We found that R. serpentina was capable of producing highly detrimental effects in our in vitro renal cell system. These results suggest more studies are needed to investigate the safety of this dietary supplement in both kidney and other target organ systems.

Phase 2 clinical trial of rapamycin-resistant donor CD4+ Th2/Th1 (T-Rapa) cells after low-intensity allogeneic hematopoietic cell transplantation.

In experimental models, ex vivo induced T-cell rapamycin resistance occurred independent of T helper 1 (Th1)/T helper 2 (Th2) differentiation and yielded allogeneic CD4(+) T cells of increased in vivo efficacy that facilitated engraftment and permitted graft-versus-tumor effects while minimizing graft-versus-host disease (GVHD). To translate these findings, we performed a phase 2 multicenter clinical trial of rapamycin-resistant donor CD4(+) Th2/Th1 (T-Rapa) cells after allogeneic-matched sibling donor hematopoietic cell transplantation (HCT) for therapy of refractory hematologic malignancy. T-Rapa cell products, which expressed a balanced Th2/Th1 phenotype, were administered as a preemptive donor lymphocyte infusion at day 14 post-HCT. After T-Rapa cell infusion, mixed donor/host chimerism rapidly converted, and there was preferential immune reconstitution with donor CD4(+) Th2 and Th1 cells relative to regulatory T cells and CD8(+) T cells. The cumulative incidence probability of acute GVHD was 20% and 40% at days 100 and 180 post-HCT, respectively. There was no transplant-related mortality. Eighteen of 40 patients (45%) remain in sustained complete remission (range of follow-up: 42-84 months). These results demonstrate the safety of this low-intensity transplant approach and the feasibility of subsequent randomized studies to compare T-Rapa cell-based therapy with standard transplantation regimens.

Phase I trial of adoptive cell transfer with mixed-profile type-I/type-II allogeneic T cells for metastatic breast cancer.

PURPOSE: Metastatic breast cancer (MBC) response to allogeneic lymphocytes requires donor T-cell engraftment and is limited by graft-versus-host disease (GVHD). In mice, type-II-polarized T cells promote engraftment and modulate GVHD, whereas type-I-polarized T cells mediate more potent graft-versus-tumor (GVT) effects. This phase I translational study evaluated adoptive transfer of ex vivo costimulated type-I/type-II (T1/T2) donor T cells with T-cell-depleted (TCD) allogeneic stem cell transplantation (AlloSCT) for MBC. EXPERIMENTAL DESIGN: Patients had received anthracycline, taxane, and antibody therapies, and been treated for metastatic disease and a human leukocyte antigen (HLA)-identical-sibling donor. Donor lymphocytes were costimulated ex vivo with anti-CD3/anti-CD28 antibody-coated magnetic beads in interleukin (IL)-2/IL-4-supplemented media. Patients received reduced intensity conditioning, donor stem cells and T1/T2 cells, and monitoring for toxicity, engraftment, GVHD, and tumor response; results were compared with historical controls, identically treated except for T1/T2 product infusions. RESULTS: Mixed type-I/type-II CD4(+) T cells predominated in T1/T2 products. Nine patients received T1/T2 cells at dose level 1 (5 × 10(6) cells/kg). T-cell donor chimerism reached 100% by a median of 28 days. Seven (78%) developed acute GVHD. At day +28, five patients had partial responses (56%) and none had MBC progression; thereafter, two patients had continued responses. Donor T-cell engraftment and tumor responses appeared faster than in historical controls, but GVHD rates were similar and responders progressed early, often following treatment of acute GVHD. CONCLUSION: Allogeneic T1/T2 cells were safely infused with TCD-AlloSCT, appeared to promote donor engraftment, and may have contributed to transient early tumor responses.

Pentostatin plus cyclophosphamide safely and effectively prevents immunotoxin immunogenicity in murine hosts.

PURPOSE: The success of immunotoxin therapy of cancer is limited by host production of neutralizing antibodies, which are directed toward the Pseudomonas exotoxin A (PE) component. In this proof-of-principle study using a well-established murine model, we hypothesized that a newly developed immune depletion regimen consisting of pentostatin plus cyclophosphamide would abrogate anti-immunotoxin reactivity. EXPERIMENTAL DESIGN: BALB/c hosts were injected weekly with recombinant immunotoxin (RIT) SS1P, which is an antimesothelin Fv antibody fragment genetically fused to a 38 kDa portion of PE, and has been evaluated in clinical trials. Experimental cohorts received induction chemotherapy consisting of pentostatin (P) plus cyclophosphamide (C) prior to initial RIT exposure; some cohorts received further maintenance PC therapy of varying intensity just prior to each weekly RIT challenge. Cohorts were monitored for T, B, myeloid cell depletion, and for total anti-SS1P antibody (Ab) formation. RESULTS: Controls uniformly developed anti-SS1P Ab after the third RIT exposure. Induction PC therapy reduced the frequency of hosts with anti-SS1P Ab. Abrogation of antibody generation was improved by maintenance PC therapy: nearly 100% of recipients of intensive PC maintenance were free of anti-SS1P Ab after 9 weekly RIT doses. The most effective PC regimen yielded the greatest degree of host B-cell depletion, moderate T-cell depletion, and minimal myeloid cell depletion. CONCLUSIONS: Induction and maintenance PC chemotherapy safely prevented anti-immunotoxin antibody formation with uniform efficacy. These data suggest that immunotoxin therapy might be used in combination with pentostatin plus cyclophosphamide chemotherapy to improve the targeted therapy of cancer.

The pentostatin plus cyclophosphamide nonmyeloablative regimen induces durable host T cell functional deficits and prevents murine marrow allograft rejection.

We describe a novel animal model of nonmyeloablative bone marrow transplantation (BMT) using the purine analog pentostatin. Other cohorts of mice received another purine analog, fludarabine, which we and others have previously evaluated in nonmyeloablative murine models. We evaluated pentostatin for its ability to (1) operate synergistically with cyclophosphamide to induce host T cell depletion; (2) induce host T cell suppression, as defined by modulation of cytokine secretion in vitro and abrogation of host-versus-graft reactivity in vivo; (3) constrain host T cell recovery post-therapy; and (4) prevent the rejection of T cell-depleted, fully major histocompatibility complex-mismatched bone marrow allografts. Relative to single-agent regimens, combination regimens with pentostatin and cyclophosphamide (PC) and with fludarabine and cyclophosphamide (FC) worked synergistically to deplete host CD4(+) and CD8(+) T cells. PC and FC regimens were developed that yielded similar levels of host T cell and myeloid cell depletion. In the setting of these generally comparable states of host T cell and myeloid cell depletion, the PC regimen was found to be highly immunosuppressive, as evidenced by a reduced host T cell capacity to secrete interleukin-2 and interferon-γ in vitro, to mediate host-versus-graft reactivity in vivo, and to recover numerically and functionally during a 2-week observation period after chemotherapy. Finally, using B6 hosts treated with the 14-day chemotherapy regimens, the PC regimen more consistently prevented the rejection of BALB/c T cell-depleted allografts compared with the FC regimen (rate of alloengraftment, 14/15 [93%] of PC-treated recipients vs 8/14 [57%] of FC-treated recipients; P < .05); similar results were observed using an 8-day conditioning regimen. These data suggest that host T cell suppression, distinct from T cell depletion, may be a critical determinant of engraftment after purine analog-based regimens and also may be preferentially attained by the use of pentostatin.

Comparative impact of trastuzumab and cyclophosphamide on HER-2-positive human breast cancer xenografts.

PURPOSE: Metronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2-positive human breast cancer models. EXPERIMENTAL DESIGN: Treatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2-positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell-secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival. RESULTS: Orthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity. CONCLUSIONS: Trastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2-positive metastatic breast cancer.

Potent induction of B- and T-cell immunity against human carcinoembryonic antigen-expressing tumors in human carcinoembryonic antigen transgenic mice mediated by direct lentivector injection.

The applicability of immunotherapy would be dramatically broadened to a greater number of recipients if direct "off-the-shelf" products could be engineered to engender functionally potent immune responses against true "self"-tumor antigens. This would obviate the need for ex vivo culture of dendritic cells or T cells on a patient-by-patient basis, for example. The carcinoembryonic antigen (CEA) is a glycoprotein expressed in normal gut epithelium that is up-regulated in the majority of colon cancers, non-small cell lung cancers, and half of all breast cancers. Such properties make CEA an excellent and important target for cancer immunotherapy. In this study, we show stabilization of 14-day established s.c. mGC4CEA tumors in human CEA (huCEA) transgenic mice following two direct low-dose injections of 0.15x10(6) transducing units of a lentiviral vector (LV) that directs expression of huCEA (LV-huCEA). This stabilization result was reproducible and detailed analyses including antibody assays, multiplex cytokine analyses on unstimulated splenocytes, lymph node cell characterizations, tetramer staining, and immunofluorescence staining of tumor sections showed that this outcome correlated with both a cellular and humoral immune response. Similar tumor outcomes were not seen when mice were vaccinated with a control LV that engineered expression of enGFP only. The long-term potency of this vaccination strategy was also studied and revealed the requirement for maintenance of tumor antigen-specific immunity for efficient tumor control. These data support the use of direct injections of low doses of LV-huCEA for enhancement of tumor immunotherapy directed against CEA.

Long-term progression and therapeutic response of visceral metastatic disease non-invasively monitored in mouse urine using beta-human choriogonadotropin secreting tumor cell lines.

Historically, the use of mouse models of metastatic disease to evaluate anticancer therapies has been hampered because of difficulties in detection and quantification of such lesions without sacrificing the mice, which in turn may also be dictated by institutional or ethical guidelines. Advancements in imaging technologies have begun to change this situation. A new method to non-invasively measure tumor burden, as yet untested to monitor spontaneous metastases, is the use of transplanted tumors expressing secretable human beta-chorionic gonadotropin (beta-hCG) that can be measured in urine. We describe examples of beta-hCG-transfected tumor cell lines for evaluating the effect of different therapies on metastatic disease, which in some cases involved monitoring tumor growth for >100 days. We used beta-hCG-tagged mouse B16 melanoma and erbB-2/Her-2-expressing human breast cancer MDA-MB-231 models, and drug treatments included metronomic low-dose cyclophosphamide chemotherapy with or without a vascular endothelial growth factor receptor 2-targeting antibody (DC101) or trastuzumab, the erbB-2/Her-2-targeting antibody. Both experimental and spontaneous metastasis models were studied; in the latter case, an increase in urine beta-hCG always foreshadowed the development of lung, liver, brain, and kidney metastases. Metastatic disease was unresponsive to DC101 or trastuzumab monotherapy treatment, as assessed by beta-hCG levels. Our results also suggest that beta-hCG levels may be set as an end point for metastasis studies, circumventing guidelines, which have often hampered the use of advanced disease models. Collectively, our data indicates that beta-hCG is an effective noninvasive preclinical marker for the long term monitoring of untreated or treated metastatic disease.

Tumor protection following vaccination with low doses of lentivirally transduced DCs expressing the self-antigen erbB2.

Gene therapy strategies may accelerate the development of prophylactic immunotherapy against cancer. We synthesized a lentiviral (LV) vector encoding a kinase-deficient form of erbB2 (erbB2tr) to transduce murine dendritic cells (DCs) efficiently. Murine erbB2 models a clinically relevant tumor-associated self-antigen; its human homolog (HER-2/neu) is overexpressed in breast cancer and in 80% of metastatic prostate cancers. Following one infection, approximately 47% of DCs overexpressed erbB2tr. To determine whether low doses of transduced DCs could protect mice from prostate cancer cells, we performed prime/boost vaccinations with 2 x 10(3) or 2 x 10(5) erbB2tr-transduced DCs. Six weeks after vaccination, mice were simultaneously bilaterally challenged with the aggressive RM-1 prostate cancer cell line and an erbB2tr-expressing variant (RM-1-erbB2tr). Whereas control mice developed both tumors, all recipients of 2 x 10(5) erbB2tr-transduced DCs developed only wild-type RM-1 tumors. One-third of mice vaccinated with just 2 x 10(3) erbB2tr-transduced DCs also demonstrated erbB2tr-specific tumor protection. Protection against RM-1-erbB2tr tumors was associated with sustained levels of anti-erbB2tr antibody production and also correlated with erbB2tr-specific Th1 cytokine secretion. Depletion of CD4(+), CD8(+), or natural killer (NK) cells prior to tumor challenge underscored their role in mediating tumor protection. We conclude that administration of DCs expressing a self-antigen through efficient LV-based gene transfer activates cellular and humoral immunity, protecting host animals against specific tumor challenge.

Lentivirally transduced recipient-derived dendritic cells serve to ex vivo expand functional FcRgamma-sufficient double-negative regulatory T cells.

alphabetaTCR(+)CD4(-)CD8(-) double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8(+) and CD4(+) T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I L(d) antigen (LV-L(d)), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-L(d)-transduced mature DCs (mDCs) were able to expand effectively both FcRgamma(-/-) and FcRgamma(+/+) DN T cells. After expansion with LV-L(d)-transduced mDCs, only the FcRgamma(+/+) DN Treg cells maintained their ability to suppress CD8(+) T cells in vitro. In addition, adoptive transfer of the FcRgamma(+/+) ex vivo expanded DN Treg cells significantly prolonged the survival of L(d+) skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRgamma(-/-) DN T cells can be expanded, they do not gain regulatory ability.

Lentivirally Transduced Recipient-derived Dendritic Cells Serve to Ex Vivo Expand Functional FcRγ-sufficient Double-negative Regulatory T Cells.

αßTCR+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8+ and CD4+ T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I Ld antigen (LV-Ld), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-Ld-transduced mature DCs (mDCs) were able to expand effectively both FcRγ-/- and FcRγ+/+ DN T cells. After expansion with LV-Ld-transduced mDCs, only the FcRγ+/+ DN Treg cells maintained their ability to suppress CD8+ T cells in vitro. In addition, adoptive transfer of the FcRγ+/+ex vivo expanded DN Treg cells significantly prolonged the survival of Ld+ skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRγ-/- DN T cells can be expanded, they do not gain regulatory ability.

Cancer immunotherapy using virally transduced dendritic cells: animal studies and human clinical trials.

The immune system uses a process known as 'immunosurveillance' to help prevent the outgrowth of tumors. In cancer immunotherapy, a major goal is for immunity against tumor-associated antigens to be generated or strengthened in patients. To achieve this goal, several approaches have been tested, including the use of highly potent antigen-presenting cells called dendritic cells (DCs), which can activate T cells efficiently. Presentation of peptides derived from tumor antigens on the surface of DCs can stimulate strong antitumor immunity. Using recombinant viral vectors encoding tumor-associated antigens, DCs can be engineered efficiently to express sustained levels of tumor-antigen peptides. This review discusses the effectiveness of virally transduced DCs in treating tumors and generating antigen-specific T-cell responses. It covers mouse and nonhuman primate studies, preclinical in vitro human cell experiments and clinical trials.

Strategies for delaying or treating in vivo acquired resistance to trastuzumab in human breast cancer xenografts.

PURPOSE: Acquired resistance to trastuzumab (Herceptin) is common in patients whose breast cancers show an initial response to the drug. The basis of this acquired resistance is unknown, hampering strategies to delay or treat such acquired resistance, due in part to the relative lack of appropriate in vivo tumorigenic models. EXPERIMENTAL DESIGN: We derived an erbB-2-positive variant called 231-H2N, obtained by gene transfection from the highly tumorigenic erbB-2/HER2-negative human breast cancer cell line, MDA-MB-231. Unlike MDA-MB-231, the 231-H2N variants was sensitive to trastuzumab both in vitro and especially in vivo, thus allowing selection of variant resistant to drug treatment in the latter situation after showing an initial response. RESULTS: The growth of established orthotopic tumors in severe combined immunodeficient mice was blocked for 1 month by trastuzumab, after which rapid growth resumed. These relapsing tumors were found to maintain resistance to trastuzumab, both in vitro and in vivo. We evaluated various therapeutic strategies for two purposes: (a) to delay such tumor relapses or (b) to treat acquired trastuzumab resistance once it has occurred. With respect to the former, a daily oral low-dose metronomic cyclophosphamide regimen was found to be particularly effective. With respect to the latter, an anti-epidermal growth factor receptor antibody (cetuximab) was effective as was the anti-vascular endothelial growth factor (anti-VEGF) antibody bevacizumab, which was likely related to elevated levels of VEGF detected in trastuzumab-resistant tumors. CONCLUSIONS: Our results provide a possible additional rationale for combined biological therapy using drugs that target both erbB-2/HER2 and VEGF and also suggest the potential value of combining less toxic metronomic chemotherapy regimens not only with targeted antiangiogenic agents but also with other types of drug such as trastuzumab.