María J. Rico

Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment

Accumulating evidence indicates that a dynamic cross-talk between tumors and the immune system can regulate tumor growth and metastasis. Increased understanding of the biochemical nature of tumor antigens and the molecular mechanisms responsible for innate and adaptive immune cell activation has revolutionized the fields of tumor immunology and immunotherapy. Both the protective effects of the immune system against tumor cells (immunosurveillance) and the evasion of tumor cells from immune attack (tumor-immune escape) have led to the concept of cancer immunoediting, a proposal which infers that a bidirectional interaction between tumor and inflammatory/regulatory cells is ultimately responsible for orchestrating the immunosuppressive network at the tumor site. In this context, a major challenge is the potentiation or redirection of tumor antigen-specific immune responses. The success in reaching this goal is highly dependent on an improved understanding of the interactions and mechanisms operating during the different phases of the cancer immunoediting process. In this review, we discuss the multiple defense and counterattack strategies that tumors have devised in order to evade immune attack and to thwart the effectiveness of several immunotherapeutic approaches.

Low-dose cyclophosphamide modulates galectin-1 expression and function in an experimental rat lymphoma model

In recent years, one of the most important insights into tumor immunity was provided by the identification of negative regulatory pathways and immune escape strategies that greatly influence the magnitude of antitumor responses. Galectin-1 (Gal-1), a member of a family of highly conserved β-galactoside-binding proteins, has been recently shown to contribute to tumor cell evasion of immune responses by modulating survival and differentiation of effector T cells. However, there is still scarce information about the regulation of Gal-1 expression and function in vivo. Here we show that administration of a single low-dose cyclophosphamide (Cy), which is capable of restraining metastasis in the rat lymphoma model L-TACB, can also influence Gal-1 expression in primary tumor, metastasis, and spleen cells and modulate the effects of this protein on T cell survival. A time-course study revealed a positive correlation between Gal-1 expression and tumor volume in primary tumor cells. Conversely, Gal-1 expression was significantly reduced in spleen cells and lymph node metastasis throughout the period studied. Interestingly, cyclophosphamide treatment was capable of restoring the basal levels of Gal-1 expression in primary tumors and spleens. In addition, this antimetastatic agent rendered spleen T cells from tumor-bearing animals resistant to Gal-1-induced cell death. Our results suggest that, in addition to other well-known functions of cyclophosphamide, this immunomodulatory agent may also modulate Gal-1 expression and function during tumor growth and metastasis with critical implications for tumor-immune escape and immunotherapy.

Therapeutic efficacy of metronomic chemotherapy with cyclophosphamide and doxorubicin on murine mammary adenocarcinomas

BACKGROUND Metronomic chemotherapy (MCT) refers to the chronic and equally spaced administration of low doses of different chemotherapy drugs, without extended rest periods. Herein, we investigated the therapeutic efficacy of metronomic cyclophosphamide (Cy) combined with doxorubicin (Dox) in two mouse mammary adenocarcinoma models. MATERIALS AND METHODS Mice were s.c. challenged with M-234p or M-406 mammary tumors, and when the tumors reached ∼150 mm(3), they were treated with: (I) no treatment (controls); (II) Cy in the drinking water (30 mg/kg body weight/day); (III) Dox (0.5 mg/kg body weight i.p. three times/week); (IV) treated as (II) + (III). Mice challenged i.v. with M-234p or M-406 tumor cells received, on day 3, the same treatments. RESULTS We found that MCT with Cy plus Dox inhibited tumor growth, decreased lung metastases, and increased the median survival time, while having low toxic effect. Combined MCT was more effective than each monotherapy causing decrease in VEGF serum concentration and tumor proliferation rate plus increase in tumor apoptosis. CONCLUSION(S) The therapeutic benefits of combined MCT with Cy and Dox on mammary adenocarcinomas together with its low toxic effect profile suggest the possibility of future translation into the clinic.

Association between baseline VEGF/sVEGFR-2 and VEGF/TSP-1 ratios and response to metronomic chemotherapy using cyclophosphamide and celecoxib in patients with advanced breast cancer

BACKGROUND Metronomic chemotherapy (MCT) with cyclophosphamide (Cy) and celecoxib (Cel) has therapeutic efficacy and low toxicity profile in advanced breast cancer patients (ABCP), but no reliable biomarkers of response have been found yet that allow patient selection for treatment. AIM To investigate the potential role as biomarkers of pro- and antiangiogenic parameters and evaluate their response in ABCP receiving metronomic Cy 50 mg p.o./day + Cel 400 mg p.o./day. MATERIALS AND METHODS Serum levels of vascular endothelial growth factor-C (VEGF-C), soluble VEGF receptors 2 and 3 (sVEGFR-2, sVEGFR-3), were measured at different time points in 13/15 patients included in a phase II trial of MCT with Cy+Cel. RESULTS Serum levels of sVEGFR-2 and sVEGFR-3 increased significantly during treatment (P = 0.0392; P = 0.0066, respectively). VEGF-C showed no significant modifications. Previous determinations of VEGF and TSP-1 in the same patients were utilized. VEGF/sVEGFR-2, VEGF/TSP-1, and VEGF-C/sVEGFR-3 ratios decreased significantly along the treatment (P = 0.0092; P = 0.0072; P = 0.0141, respectively). Nonsignificant variations were observed for VEGF-C/sVEGFR-2 ratio. Baseline values of VEGF/sVEGFR-2 and VEGF/TSP-1 ratios were associated with time to progression (TTP) (P = 0.0407; P = 0.0394, respectively) meanwhile baseline VEGF was marginally significant (P = 0.0716). Patients with values lower than the 50th percentile for both ratios showed longer TTP. CONCLUSIONS We have identified the baseline VEGF/sVEGFR-2 and VEGF/TSP-1 ratios as potential biomarkers of response in ABCP treated metronomically with Cy+Cel. This finding warrants its confirmation in a higher number of patients.

Comparative Effectiveness of Two Metronomic Chemotherapy Schedules—Our Experience in the Preclinical Field

Metronomic chemotherapy refers to the chronic, equally spaced, delivery of low doses of chemotherapeutic drugs, without extended interruptions. Previously, we developed two combined metronomic schemes for the treatment of murine mammary tumors. The aim of this study was to compare their effects on tumor and metastasis growth, survival, and toxicity. Metronomic chemotherapy with Cyclophosphamide + Celecoxib (Cy + Cel) showed higher antimetastatic power than Cyclophosphamide + Doxorubicin (Cy + Dox), while being similar in other aspects. That difference, plus the advantage that represents its oral administration, suggests that the Cy + Cel combination is more suitable than Cy + Dox for metronomic chemotherapy of mammary tumors and could be proposed to the translation to the clinic.

Putative Biomarkers of Response to Treatment in Breast Cancer Patients: A Pilot Assay

ABSTRACT Identifying tumor biomarkers associated with clinical behavior in breast cancer patients may allow higher accuracy in the selection of treatment. Different types of cells were determined in the primary tumors of stage I, II, and III of breast cancer patients, who were assigned to one of the two groups: (1) disease-free or (2) relapsed/progressed, at 5 years after primary treatment. We studied 32 tumor samples. CD4+ lymphocytes and CD44+CD24−/low cells (cancer stem cells) showed a significant association with clinical outcome at 5 years of primary treatment, while CD8+, Foxp3+, CD34+, and myeloid-derived suppressor cells did not show any association. Coincident with the results of individual analysis, we identified CD4+ cells and CD44+CD24−/low cells as good predictors of long-term clinical outcome in a logistic regression.

Abstract P3-07-61: Predictors of response and follow up biomarkers for metronomic chemotherapy with cyclophosphamide and celecoxib in advanced breast cancer patients

Low-dose metronomic chemotherapy (MC) with Cyclophosphamide (Cy) and Celecoxib (Cel) has demonstrated to be effective and well-tolerated in advanced breast cancer patients (ABCP) but predictive markers of response or follow-up are lacking. Given the antiangiogenic properties of MC we analyzed several angiogenesis-related biomarkers and evaluated their potential role as predictors of response or treatment follow-up of ABCP treated with MC. Treatment plan: Patients received Cy 50 mg p.o./day + Cel 200 mg p.o./ bid. Cellular parameters: Circulating endothelial cells (CEC) and Circulating progenitor endothelial Cells (CEP) were determined by Flow Cytometry. Serologic parameters: Serum levels of vascular endothelial growth factor (VEGF), VEGF-C, soluble VEGF Receptors 2 and 3 (sVEGFR-2, sVEGFR-3) and Thrombospondin-1 (TSP-1) were determined by ELISA. Blood samples were collected before and during treatment. Twenty patients were enrolled. Response Rate was 5% and Clinical Benefit (CB) 55%. Most of the patients showed prolonged stable disease (SD≥24 weeks). Biomarkers were determined in all patients. Levels of CEC and CEP showed no clear trend variations during treatment. However, levels of CEC significantly increased at the time of disease progression in those patients who showed CB (P=0.014). Also baseline values of CEC and CEP showed marginally significant associations withTime To Progression. Serum VEGF concentration decreased during treatment (P=0.050) while sVEGFR-2 increased (P=0.005). VEGF-C, sVEGFR-3 and TSP-1 showed non-significant variations. VEGF/sVEGFR-2 ratio decreased during treatment (P=0.041), whereas VEGF/TSP-1, and VEGF-C/sVEGFR-2 ratios showed non-significant variations. Baseline values of VEGF, and VEGF/sVEGFR-2 showed negative and significant associations with TTP (P=0.0354 and P=0.0300, respectively) while sVEGFR-2 did not. When considering the two variables together, the goodness of prediction was not improved. To confirm the value of baseline VEGF and VEGF/sVEGFR-2 as predictors of response, we used the 50th percentile as a cutoff value to analyze the % of progression free survival. Patients with values lower than the 50th percentile for both biomarkers showed longer TTP (P=0.0001 and P=0.0008, respectively). The treatment had anti-angiogenic effect (VEGF decrease and sVEGFR-2 increase). The absence of variation in VEGF-C and sVEGFR-3 would indicate the lack of effect on lymphangiogenesis. Increased levels of CEC could be useful for detecting progression. If confirmed with a higher number of patients, baseline VEGF and VEGF/sVEGFR-2 values could be useful as early predictors of response. Citation Format: Perroud HA, Alasino CM, Rico MJ, Menacho-Marquez MA, Mainetti LE, Queralt F, Pezzotto SM, Rozados VR, Scharovsky G. Predictors of response and follow up biomarkers for metronomic chemotherapy with cyclophosphamide and celecoxib in advanced breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-61.

Abstract 795: Metronomic chemotherapy with cyclophosphamide and metformin inhibits tumor and metastasis growth M-406 murine mammary adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of different chemotherapeutic drugs, without extended interruptions. We have already demonstrated that MCT, as an intervention strategy, of metronomic Cy combined with celecoxib or doxorubicin inhibits mammary tumors growth. Our present aim was to analyze the antitumor and antimetastatic effects of MCT of Cy combined with Met. Inbred CBi mice were s.c. or i.v. challenged with M-406 tumor (Day 0), and treated when the tumors reached a volume of 100 a 150 mm3 (s.c.) or from Day 3 (i.v.) on, with: I) no further treatment (controls); II) Cy in the drinking water (20-30 mg/kg body weight/day); III) Met 3 times/week by gavage (100 mg/kg); IV) Treated as II + III. Tumors were measured and animals weighed twice a week and blood samples for glycemia determination were taken. On day 36 tumor volume in group IV (mm3, mean ± SEM: 929.7 ± 213.8) was lower than in groups I (2880.0 ± 1530.0), II (2896.4 ± 976.9) and III (3772.0 ± 68.0). On day 46, with only two groups remaining, tumor volume in group IV was lower than that in group II (p=0.032). All the groups differed in tumor volume doubling time (days, mean ± SEM; I: 3.8 ± 0.3, II: 5.3 ± 0.7, III: 3.6 ± 0.4, IV: 5.4 ± 0.2) (p<0.05). All the groups differed in survival (days, median, I: 30.5; II: 44; III: 29; IV: 50) (p=0.0063), showing group IV the highest one. There were neither weight losses nor changes in the glycemia with respect to basal values both in s.c. and i.v. experiments. In the i.v. experiment, animals were sacrificed on day 13, when the first mouse showed signs of metastatic illness. The number and diameter of lung metastasis were determined and total metastatic volume (TMV) was calculated. The experimental groups differed in TMV (p<0.0001), being those of groups II (mm3, mean ± SEM: 162.1 ± 41.4) and IV (218.6 ± 31.3) significantly lower (p<0.001 and p<0.01, respectively) than those in groups I (491.3 ± 80.6) and III (432.3 ± 54.5). These results clearly show that metronomic chemotherapy with a combination of cyclophosphamide and metformin significantly inhibits tumor growth and increase survival, also decreasing the development of metastasis of a murine mammary adenocarcinoma, while being devoid of toxicity. Citation Format: Antonela S. Asad, Jesus Basualdo, Lucia Micheletti, Maria C. Capello Gardenal, Herman A. Perroud, Maria J. Rico, Viviana R. Rozados, O. Graciela Scharovsky. Metronomic chemotherapy with cyclophosphamide and metformin inhibits tumor and metastasis growth M-406 murine mammary adenocarcinoma. [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 795. doi:10.1158/1538-7445.AM2014-795

Abstract 4665: VEGF/SVEGFR-2 and VEGF/TSP-1 ratios as probable predictors of response to metronomic chemotherapy with Cyclophosphamide and Celecoxib in advanced breast cancer patients.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The efficacy and low toxicity of antiangiogenic metronomic chemotherapy (MCT) with cyclophosphamide (Cy) and celecoxib (Cel) in advanced breast cancer patients (ABCP) was demonstrated in our studies but no reliable biomarkers or predictors of response have been found yet. The aim of this study was to analyze several pro- and anti-angiogenic parameters and evaluate their potential role as predictor of response duration in ABCP treated with MCT with daily Cy and Cel. Treatment plan: Patients received Cy 50 mg p.o./day + Cel 200 mg p.o. bid. This clinical trial was authorized by the School of Medicine Bioethics Committee and by A.N.M.A.T. (Argentine Regulatory Authority). .Angiogenesis parameters: Serum levels of vascular endothelial growth factor (VEGF), VEGF-C, soluble VEGF Receptors 2 and 3 (sVEGFR-2, sVEGFR-3) and Thrombospondin 1 (TSP-1) were determined by ELISA. Blood samples were collected before and during treatment and parametric tests were used to analyze data. Fifteen patients were enrolled, A partial response (PR) was observed in 1 patient (6.7%), which lasted 6 weeks. Prolonged Stable disease (pSD ≥24 weeks) was observed in 6/15 patients (40%). Median TTP among patients with pSD was 37.5 weeks (range: 26.43-81.57). The OCB obtained was 46.7% (PR=1/15 + SD=6/15). The median TTP (PR+pSD) was 33 weeks (range: 10.43-81.57). Treatment toxicity was very low and no grade 3 or 4 adverse events were registered. Biomarkers were determinated in 13 patients. Serum VEGF concentration decreased as a function of time (P=0.004); sVEGFR-2 increased during response (P= 0.0268) while non-significant variations were detected in VEGF-C, sVEGFR-3 and TSP-1. The baseline values of VEGF, and VEGF/sVEGFR-2 and VEGF/TSP-1 ratios were significantly correlated with response duration (P=0.029, P=0.015, P=0.014, respectively). To assess which of the three variables measured at the beginning of the treatment could be used to predict response duration, linear regression analyses were done. VEGF/sVEGFR-2 and VEGF/TSP-1 ratios were good predictors (P=0.028, P=0.029, respectively), meanwhile VEGF was not (P=0.059). When considering VEGF, VEGF/VEGFR-2 and VEGF/TSP-1 in a multiple regression analysis, the goodness of prediction was not improved with respect to that obtained with each putative predictor. We conclude that the antiangiogenic nature of MCT with Cy plus Cel is confirmed through the decrease of VEGF and the increase of sVEGFR-2; the absence of variation in VEGF-C and sVEGFR-3 would indicate the lack of effect on lymphangiogenesis; if confirmed in a higher number of patients, VEGF/sVEGFR-2 and VEGF/TSP-1 ratios could be useful as early predictors of treatment response. Citation Format: Herman A. Perroud, Carlos M. Alasino, Maria J. Rico, Francisco Queralt, Stella M. Pezzotto, Viviana R. Rozados, O.Graciela Scharovsky. VEGF/SVEGFR-2 and VEGF/TSP-1 ratios as probable predictors of response to metronomic chemotherapy with Cyclophosphamide and Celecoxib in advanced breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4665. doi:10.1158/1538-7445.AM2013-4665

Abstract 1749: Phase I/II clinical trial: Metronomic chemotherapy with cyclophosphamide (Cy) and celecoxib (Cel) in breast cancer patients progressing after standard chemotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Metronomic chemotherapy (MCT), the chronic administration at regular intervals of low doses of chemotherapeutic drugs without extended rest periods, allows chronic treatment with therapeutic efficacy and low toxicity. Its molecular target is mainly the endothelial cell which is less prone to develop drug resistance. The aim of the present two-stage, phase I/II trial study was to determine the safety and efficacy of MCT with Cy plus Cel in metastatic breast cancer (MBC) patients progressing after standard chemotherapy and to identify early markers of therapeutic response. Patient inclusion criteria: 21-80 years old, more than 3 month of life expectancy, advanced breast cancer in progression after, at least, anthracyclines, taxanes and capecitabine treatments, at least one lesion according to RECIST criteria, ECOG scale β2, adequate bone marrow, hepatic, and renal function, normal calcemia, signed informed consent. Treatment: CY, 50 mg p.o./day + CEL, 200 mg p.o. bid. Study design: The trial has two stages with a final number of 25 patients, looking for a 25% general response rate (≥ error=0.10; ≤ error=0.05). Primary end point: clinical response, safety and tolerability. Toxicity: all the serious adverse events are registered by CTCAE criteria and followed until their resolution. Angiogenesis parameters: the serum level of vascular endothelial growth factor (VEGF) and thrombospondin 1 (TSP-1) were determined by ELISA, and the circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs) by flow cytometry. This protocol was authorized by the School of Medicine Bioethics Committee and by A.N.M.A.T. (National Regulatory Authority). During the first stage we have incorporated 13 patients. The time of permanence varied from 4 to 64 weeks (median=13). Presently, 4 patients are still in treatment. It was observed stable disease (SD) in 9/13 patients that lasted from 12 to 64 weeks (median=18) and partial response (PR) in 1/13. Treatment toxicity was very low: hematologic (2/13, grade I and II), gastric (4/13, grade I). There was no evidence of hepatic, renal or cardiac toxicities associated with the therapy. The performance status evaluated with the ECOG scale showed no modifications in 2/10 patients, a worse score in 3/10 and a better one in 5/10. The % of circulating CEPs showed a significant increase in non-responder patients (p=0.008) The serum VEGF concentration decreased as a function of time (p=0.004). Patients with the lowest VEGF/TSP-1 ratios showed the highest permanence. We conclude that the treatment showed a low toxicity profile, the therapeutic response consisted of SD during different periods of time, and one PR with no modifications or improvement of the performance status in a high proportion of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1749. doi:1538-7445.AM2012-1749