Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.

The HER2 receptor, overexpressed in 20-25% of breast invasive tumors, is associated with low disease-free survival. Trastuzumab (Tz), monoclonal antibody anti HER2 is used as immunotherapy in HER2+ mammary tumors; however, 60% of patients are unresponsive to this therapy. The aim of our study is the analysis of the effect of Tz on the growth of human mammary tumor cells cultured in 3D (shperoids). The human adenocarcinoma cell line BT474 (HER2+) was used to generate spheroids grown in liquid RPMI1640 medium. One spheroid/well was seeded on a 1.5% agar layer, reaching 2.5 mm diameter at day 62 of culture. Differential expression of HER2, HIF-1, Bcl-2 and Ki67 were detected by immunohistochemistry. Proliferating, quiescent as well as apoptotic cells towards the hypoxic core of the tumor spheroids were identified. The effect of Tz (10 and 50 ug/ml/spheroid, 3 times/week) on the growth of 3D cultures of BT474 cells was evaluated, beginning with a volume of 0.23 ± 0.03 mm3 (day 0). Tz 10 ug/ml arrested spheroids growth since day 7 (3 doses) while Tz 50 ug/ml induced a significant reduction in the volume since the first dose (day 0) until day 27, when spheroids showed 71% of growth inhibition compared to untreated or human IgG controls (Tz 50= 0.12 ± 0.04 mm3 vs control= 0.45 ± 0.03 mm3, p<0.001). Spheroids showed lower sensitivity to the cytostatic effect of Tz 10 ug/ml when compared to the same cells cultured as monolayers. Tz exerted a direct and dose-dependent effect on BT474 tumor spheroids: while it was cytostatic at the lower dose (Tz10), it was cytotoxic at the higher (Tz50). Nitric oxide (NO) levels measured by Griess reaction in tumor spheroid supernatants were significantly higher upon Tz50 treatment compared to untreated controls (p<0.01), suggesting NO is involved in the direct cytotoxic mechanism of Tz. BT474 cells growing in monolayers responded to TZ with higher NO levels than 3D cultures. In order to assess whether immune system and tissue architecture have an impact on the Tz cytotoxic effect, we co-cultured single BT474 tumor spheroids with macrophages from human peripheral blood (ratio1:5). At 10 days co-culture, the macrophages had a type I response (M1) against tumor cells. In fact, we observed that the presence of macrophages in the culture increased the cytotoxicity of Tz. This period of time was not enough to switch macrophages to the M2 phenotype. Conditioned media from Tz50-treated BT474 spheroids decreased MMP9 production by macrophages cultures. We propose this 3D culture model as a useful tool to study susceptibility to trastuzumab therapy in HER2 + tumor cells, as well as to analyze the mechanisms of action and resistance of this treatment. 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 2881. doi:1538-7445.AM2012-2881

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Objectives To determine the comparative efficacy and safety of a fixed dose of benznidazole (BZN) with an adjusted‐dose for Trypanosoma cruzi‐seropositive adults without cardiomyopathy. Methods We conducted a systematic review and individual participant data (IPD) meta‐analysis following Cochrane methods, and the PRISMA‐IPD statement for reporting. Randomised controlled trials (RCTs) allocating participants to fixed or adjusted doses of BZN for T. cruzi‐seropositive adults without cardiomyopathy were included. We searched (December 2021) Cochrane, MEDLINE, EMBASE, LILACS and trial registries and contacted Chagas experts. Selection, data extraction, risk of bias assessment using the Cochrane tool, and a GRADE summary of finding tables were performed independently by pairs of reviewers. We conducted a random‐effects IPD meta‐analysis using the one‐stage strategy, or, if that was impossible, the two‐stage strategy. Results Five RCTs (1198 patients) were included, none directly comparing fixed with adjusted doses of BZN. Compared to placebo, BZN therapy was strongly associated with negative qPCR and sustainable parasitological clearance regardless of the type of dose and subgroup analysed. For negative qPCR, the fixed/adjusted rate of odds ratios (RORF/A) was 8.83 (95% CI 1.02–76.48); for sustained parasitological clearance, it was 4.60 (95% CI 0.40–52.51), probably indicating at least non‐inferior effect of fixed doses, with no statistically significant interactions by scheme for global and most subgroup estimations. The RORF/A for treatment interruption due to adverse events was 0.44 (95% CI 0.14–1.38), probably indicating no worse tolerance of fixed doses. Conclusions We found no direct comparison between fixed and adjusted doses of BZN. However, fixed doses versus placebo are probably not inferior to weight‐adjusted doses of BZN versus placebo in terms of parasitological efficacy and safety. Network IPD meta‐analysis, through indirect comparisons, may well provide the best possible answers in the near future. Registration The study protocol was registered in PROSPERO (CRD42019120905).