Background:Although there are reports that metronomic cyclophosphamide (CTX) can be immune stimulating, the impact of its combination with anti-CTLA-4 immunotherapy for the treatment of cancer remains to be evaluated.Methods:Murine EMT-6/P breast cancer, or its cisplatin or CTX-resistant variants, or CT-26 colon, were implanted into Balb/c mice. Established tumours were monitored for relative growth following treatment with anti-CTLA-4 antibody alone or in combination with; (a) metronomic CTX (ldCTX; 20 mg kg−1 day−1), b) bolus (150 mg kg−1) plus ldCTX, or (c) sequential treatment with gemcitabine (160 mg kg−1 every 3 days).Results:EMT-6/P tumours responded to anti-CTLA-4 therapy, but this response was less effective when combined with bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with regimens of either sequential or concomitant gemcitabine, including in orthotopic EMT-6 tumours, and independently of the schedule of drug administration. Tumour responses were confirmed with CT-26 tumours but were less pronounced in drug-resistant EMT-6/CTX or EMT-6/DDP tumour models than in the parent tumour. A number of tumour bearing mice developed spontaneous metastases under continuous therapy. The majority of cured mice rejected tumour re-challenges.Conclusions:Metronomic CTX can be combined with anti-CTLA-4 therapy, but this therapy is impaired by concomitant bolus CTX. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in chemotherapy-naive tumours, although tumour relapses can occur, in some cases accompanied by the development of spontaneous metastases.

Although a number of Her-2 targeting drugs are now available for the treatment of Her-2 positive breast cancers, resistance to these therapies rapidly emerges. There is therefore a need to study how Her-2 targeting strategies work in vivo, and the mechanisms by which tumors eventually become resistant to anti-Her-2 therapies. We report the in vivo selection of variants of the human Her-2 positive breast cancer cell lines BT474 and MDA-MB361. Both cell lines were initially implanted orthotopically into Severely Compromised Immunodeficient mice, and the resulting tumors were serially passaged into new hosts over a 4 year period. This selection process produced the BT474V3 and MDA361V3 variants, which grow rapidly in vivo and which retain their Her-2 over-expression, and which respond (p<0.05, relative to controls) to trastuzumab (20mg/kg i.p. every 3 days). Tumor variants were also derived from selected tumors that relapsed after 3 months of continuous trastuzumab monotherapy. MDA361V3 tumors showed a significant (p<0.05) response to the combination therapy of trastuzumab plus metronomic cyclophosphamide (20mg/kg/day, p.o.). Tumors eventually progressed under this combination therapy, and cell lines were derived from the drug resistant tumors. Non-invasive monitoring of the Her-2 positive models in vivo was achieved by luciferase transfection of the tumor variants, or by transfection of chorionic gonadotropin (hCG) cDNA which allows for relative tumor growth to be evaluated via the resulting hCG levels in the mouse urine. Luciferase transfected BT474V3 were implanted intracranially (coordinates for implantation from Bregma were AP, +1.1; LM, -2.0; and DV, -3), and bioluminescence was detectable 20 days later. The implanted tumors in the brain eventually showed a doubling of bioluminescence every 2 weeks, which provides ample time for therapeutic intervention in this model of Her-2 positive breast cancer brain metastasis. To further model the response of Her-2 positive metastatic breast cancer, we employed our met2.hCG model, derived from the human MDA-MB-231 human breast cancer cell line. Thus, MDA-MB-231 were transduced to overexpress Her-2, then tagged with hCG, and then selected for high spontaneous metastatic capacity. Orthotopically implanted met2.hCG tumors responded to trastuzumab as a monotherapy, and when given in combination with metronomic cyclophosphamide. Following surgical removal of the orthotopically implanted met2.hCG tumors, urine hCG levels indicated that the metastases also responded (p<0.05, compared to saline treated controls) to the combination of metronomic cyclophosphamide plus trastuzumab, but they did not respond to the trastuzumab monotherapy. Collectively, our data shows that we have derived models of Her-2 positive breast cancer that can be used to evaluate anti-Her-2 therapeutic strategies, and to study the emergence of resistance to anti-Her-2 based therapies. Citation Format: Paloma A. Valenzuela, Sarah N. Jallad, Karla Parra, Natzidielly Lerma, Irving Miramontes, Alejandra Gallegos, Ping Xu, William Cruz-Munoz, Shan Man, Robert S. Kerbel, Giulio Francia. Derivation and analysis of preclinical models of human her-2 positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2014-3142

The targeting of the CTLA-4 protein with the antibody ipilimumab has been a success in terms of producing an increase in the survival of patients with unresectable melanoma, and clinical trials are ongoing to evaluate this strategy in other tumor types. Our aim in this study was to evaluate the combination of CTLA-4 blocking with metronomic chemotherapy regimens. To that end, we subcutaneously implanted murine EMT-6 breast tumor cells into syngeneic Balb/c mice (n=6-8/group) and evaluated therapies on the established tumors. Murine CTLA-4 blocking was achieved using anti-mouse CD152 (CTLA-4), clone 9H10, injected on day 1 (100ug/mouse) and on day 6 (35ug/mouse) of therapy. Anti-CTLA-4 therapy was administered on its own or combined with metronomic regimens. These included; a) Bolus (150mg/kg, i.p.) cyclophosphamide (CTX) followed by metronomic CTX (20mg/kg/day, p.o.), b) metronomic CTX, and c) sequential gemcitabine therapy (160mg/kg every 3 days, i.p.) given to the tumors relapsing after the anti-CTLA-4 therapy. We observed that control (saline) treated tumors, or tumors treated with Bolus CTX plus metronomic CTX, grew rapidly and had to be sacrificed 4 weeks after tumor implantation. Anti-CTLA-4 monotherapy produced an initial tumor regression followed by tumor relapses, 2-3 weeks later, in 5/6 mice. Surprisingly, the Bolus CTX plus metronomic CTX hindered the effective CTLA-4 therapy, failed to produce tumor regression, and resulted in rapidly growing tumors. The combination of anti-CTLA-4 plus metronomic CTX also produced tumor regression and resulted in a longer delay in the appearance of relapsing tumors (p<0.05 compared to anti-CTLA-4 alone), which also eventually appeared in 5/6 mice. A Kaplan Meier plot showed that the anti-CTLA-4 plus metronomic CTX regimen significantly improved survival compared to the anti-CTLA-4 monotherapy (p<0.05). The regimen involving first line anti-CTLA4 therapy followed by a second line gemcitabine therapy, produced a sustained tumor regression that continued for over 100 days. In this group, 5/7 mice did not show a tumor regrowth; 1 mouse showed a tumor regrowth under continuous gemcitabine therapy with concomitant development of lung metastasis. Tumor cells lines were derived from the relapsing tumor and from the lung metastasis. Collectively our data shows that Bolus plus metronomic CTX may compromise anti-CTLA-4 therapy. Furthermore, anti-CTLA-4 therapy may be effectively combined with metronomic CTX, or with a sequential gemcitabine therapy, in a preclinical model of breast cancer. Citation Format: Karla Parra, Chantal Vidal, Paloma Valenzuela, Sarah Jallad, Georgialina Rodriguez, Mitchell S. Felder, Natzidielly Lerma, Guido Bocci, Urban Emmenegger, Robert A. Kirken, Giulio Francia. Evaluation of CTLA-4 blockage with sequential metronomic chemotherapy for the treatment of preclinical breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2987. doi:10.1158/1538-7445.AM2014-2987

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