Karla Parra

Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth

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.

Resistance to metronomic chemotherapy and ways to overcome it

Therapeutic resistance is amongst the major determinants of cancer mortality. Contrary to initial expectations, antivascular therapies are equally prone to inherent or acquired resistance as other cancer treatment modalities. However, studies into resistance to vascular endothelial growth factor pathway inhibitors revealed distinct mechanisms of resistance compared to conventional cytotoxic therapy. While some of these novel mechanisms of resistance also appear to be functional regarding metronomic chemotherapy, herein we summarize available evidence for mechanisms of resistance specifically described in the context of metronomic chemotherapy. Numerous preclinically identified molecular targets and pathways represent promising avenues to overcome resistance and enhance the benefits achieved with metronomic chemotherapy eventually. However, there are considerable challenges to clinically translate the preclinical findings.

Label-Free Raman Imaging to Monitor Breast Tumor Signatures

Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for further label-free optical tools to diagnose the disease.

Abstract 2987: Evaluation of CTLA-4 blockage with sequential metronomic chemotherapy for the treatment of preclinical breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Abstract 3142: Derivation and analysis of preclinical models of human her-2 positive breast cancer

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 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