Abstract
Neutrophils play an important role in cancer progression and both pro- and antitumorigenic functions have been described. However, their role in response to therapy and whether environmental ...signals modulate their function in the tumor microenvironment (TME) remain unclear. Here, we show that neutrophil content in the TME defines the response to chemo- and immunotherapy. Neutrophil depletion or recruitment blockade impaired the response, while overexpression of neutrophil-attracting chemokines rendered non-responder tumors susceptible to therapy. Importantly, we demonstrate that tumor neutrophils are a heterogeneous and dynamic population that differ from those found in blood or bone marrow, and we identified a neutrophil subset that correlates with therapy response. Additionally, we found that mice lacking microbiota have impaired neutrophil recruitment and function associated with reduced therapy efficacy. Mechanistically, administration of a microbiota-derived NOD2 ligand to mice lacking microbiota was sufficient to restore neutrophil dynamics and response to therapy. We confirmed tumor neutrophil heterogeneity in cancer patients and showed that while total neutrophil content had no predictive value for patient’s overall survival, two specific neutrophil subsets associated with better outcome for pancreatic cancer patients. Remarkably, the subset enriched in non-responder germ-free tumors in our preclinical studies associated with a similar poor outcome in colorectal cancer patients. Collectively, our findings highlight the importance of characterizing neutrophil heterogeneity in the TME and the contribution of microbiota in shaping these cells to predict a successful therapy outcome.
Supported by NIH Intramural Program
The mammary gland hosts a microbiota, which differs between malignant versus normal tissue. We found that aerosolized antibiotics decrease murine mammary tumor growth and strongly limit lung ...metastasis. Oral absorbable antibiotics also reduced mammary tumors. In ampicillin-treated nodules, the immune microenvironment consisted of an M1 profile and improved T cell/macrophage infiltration. In these tumors, we noted an under-representation of microbial recognition and complement pathways, supported by TLR2/TLR7 protein and C3-fragment deposition reduction. By 16S rRNA gene profiling, we observed increased Staphylococcus levels in untreated tumors, among which we isolated Staphylococcus epidermidis, which had potent inflammatory activity and increased Tregs. Conversely, oral ampicillin lowered Staphylococcus epidermidis in mammary tumors and expanded bacteria promoting an M1 phenotype and reducing MDSCs and tumor growth. Ampicillin/paclitaxel combination improved the chemotherapeutic efficacy. Notably, an Amp-like signature, based on genes differentially expressed in murine tumors, identified breast cancer patients with better prognosis and high immune infiltration that correlated with a bacteria response signature.
This study highlights the significant influence of mammary tumor microbiota on local immune status and the relevance of its treatment with antibiotics, in combination with breast cancer therapies.
•Ampicillin reduces murine mammary tumors growth by perturbing the tumor microbiota.•Inflammation-promoting bacteria drop and anti-tumor immunostimulating species arise.•Oral ampicillin strongly improves paclitaxel chemotherapy efficacy.•Amp-like signature identifies good prognosis in breast cancers patients.•Antibiotics emerge as an unexpected tool to guide antitumor immune microenvironment.
Abstract
Although the clinical benefit of trastuzumab for the management of HER2-positive breast carcinomas (BCs) has been largely demonstrated, many women, even those reported to have tumors ...potentially most sensitive to trastuzumab (e.g., HER2-enriched by PAM50 and with infiltrating immune cells) do not respond to this agent. The relevance of immunity in the cytotoxic mechanism of action of trastuzumab supports the notion that the anti-tumor effect of this monoclonal antibody depends on host immune system activity. Since gut commensal bacteria reportedly contribute to the development and maintenance of the immune system and have immunomodulatory effects, we investigated whether a relationship between gut microbiota composition and the response to trastuzumab exists in patients with HER2-positive BC.
Stool samples were collected from 18 patients with primary HER2-positive BCs before the outset of neoadjuvant trastuzumab-based chemotherapy and analyzed by 16S rRNA gene profiling using Illumina Miseq platform. Gut microbiota β-diversity analysis by UniFrac algorithm revealed a heterogeneous microbiota composition mainly due to differences in the relative abundance of Bacteroides, Faecalibacterium and a genus belonging to Ruminococcaceae family. Unsupervised analysis identified two microbiota clusters that significantly discriminated patients according to pathological complete response (pCR) (p=0.0128, by Fisher test). No association between microbiota clusters and PAM50 molecular classification of tumor biopsies, as evaluated by gene expression, was observed. Moreover, HER2-enriched cases were distributed in the two microbiota clusters based on trastuzumab response. Differences in the intestinal microbiota between responsive (R) and non-responsive (NR) patients were assessed by LEfSe analysis: a significant higher and lower abundance of Clostridiales taxonomic order and Bacteroides taxonomic genus, respectively, was foundin R as compared to NR patients. Immune genes expressed in tumor biopsies that were found significantly correlated with these bacteria mainly belong to innate and adaptive immune response and cellular response to tumor necrosis factor pathways.
To investigate the causal role of gut microbiota in trastuzumab benefit, FVB mice bearing syngeneic mammary carcinoma overexpressing human HER2 were transplanted with fecal material from R and NR patients after intestinal flora depletion by the use of an antibiotic cocktail. The mouse response to trastuzumab treatment recapitulated the response observed in patients from which stool derived.
The obtained results support the contribution of gut microbiota in trastuzumab activity by influencing tumor immune microenvironment, independently of tumor intrinsic molecular characteristics. These data represent the proof-of-concept that manipulating gut microbes in resistant patients could be a new strategy to improve the response to trastuzumab.
Supported by AIRC
Citation Format: Elda Tagliabue, Martina Di Modica, Giorgio Gargari, Viola Regondi, Arianna Bonizzi, Stefania Arioli, Fabio Corsi, Simone Guglielmetti, Tiziana Triulzi. Commensal gut microbiota influences efficacy of trastuzumab in patients with HER2-positive breast carcinoma abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-32.
Hepatocellular carcinoma (HCC) is one of the common malignancies and is an increasingly important cause of cancer death worldwide. Surgery, chemotherapy, and radiation therapy extend the 5-year ...survival limit in HCC patients by only 6%. Therefore, there is a need to develop new therapeutic approaches for the treatment of this disease. The orally bioavailable proteasome inhibitor MLN2238 (ixazomib) has been demonstrated to have anticancer activity. In the present study, we investigated the preclinical therapeutic efficacy of MLN2238 in HCC cells through in vitro and in vivo models, and examined its molecular mechanisms of action. MLN2238 inhibited cell viability in human HCC cells HepG2, Hep3B, and SNU475 in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that MLN2238 induced G2/M cell cycle arrest and cellular apoptosis in HCC cells. Cell cycle arrest was associated with increased expression levels of p21 and p27. MLN2238-induced apoptosis was confirmed by caspase-3/7 activation, PARP cleavage and caspase-dependent β-catenin degradation. In addition, MLN2238 activated ER stress genes in HCC cells and increased the expression of the stress-inducible gene nuclear protein-1. Furthermore, MLN2238 treatment induced upregulation of myeloid cell leukemia-1 (Mcl-1) protein, and Mcl-1 knockdown sensitized HCC cells to MLN2238 treatment, suggesting the contribution of Mcl-1 expression to MLN2238 resistance. This result was also confirmed using the novel Mcl-1 small molecule inhibitor A1210477. Association of A1210477 and MLN2238 determined synergistic antitumor effects in HCC cells. Finally, in vivo orally administered MLN2238 suppressed tumor growth of Hep3B cells in xenograft models in nude mice. In conclusion, our results offer hope for a new therapeutic opportunity in the treatment of HCC patients.
Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and ...patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.
Abstract
Recently, the composition of the gut microbiota, due to its influence on host immune system, has been linked to the effectiveness of chemotherapy and immunotherapy. Since trastuzumab, ...besides inhibiting the HER2 signaling, recruits innate and adaptive immune cells that mediate its cytotoxic activity in the tumor, we hypothesized that commensal bacteria can be a source of heterogeneity for the response to therapy in patients with HER2-positive breast cancer (HER2+BC).
The impact of the gut microbiota on anti-HER2 therapy was studied in mice with the intestinal flora alterated by the treatment with vancomycin or streptomycin-two broad spectrum antibiotics poorly absorbed in the intestine. The association between commensal bacteria composition and clinical efficacy of trastuzumab was investigated in a cohort of HER2+BC patients treated with neoadjuvant trastuzumab.
Administration of antibiotics impaired the efficacy of anti-HER2 monoclonal antibodies both in FVB and BALB/c mice bearing syngeneic mammary carcinomas expressing HER2. 16S rRNA gene profiling of FVB mouse feces showed that both antibiotics decreased bacterial α-diversity in the gut as evaluated by Chao1, Simpson and Shannon indices, lowering the abundance of Clostridiales bacteria. Mice transplanted with feces from antibiotic treated mice did not benefit from the anti-HER2 treatment supporting a direct relation between intestinal bacteria and therapeutic efficacy. Analysis by flow cytometry and immunohistochemistry of tumors grown in mice showed that alteration of gut microbiota compromised the recruitment of CD4+ T cells and Natural Killer (CD49b+, GZMB+) cells upon anti-HER2 administration. Fecal 16S rRNA gene sequencing demonstrated a significantly higher microbial α-diversity in patients who achieved a pathological complete response compared to non-responders using several indices. Moreover, a clustering effect by patient’s response was observed visualizing the β-diversity. OTUs belonging to the Clostridiales and Bacteroidales orders were reduced and enriched, respectively, in non-responders.
Our data support that the composition of the gut microbiota, especially as regards the abundance of Clostridiales bacteria, has a role in the therapeutic efficacy of trastuzumab both in mice and patients. Therefore, the manipulation of intestinal bacteria may represent a new strategy to improve the cure of HER2+BC patients.
(Supported by Associazione Italiana per la Ricerca sul Cancro).
Citation Format: Martina Di Modica, Viola Regondi, Giorgio Gargari, Arianna Bonizzi, Stefania Arioli, Beatrice Belmonte, Claudio Tripodo, Simone Guglielmetti, Fabio Corsi, Tiziana Triulzi, Elda Tagliabue. The gut microbiota contributes to the effectiveness of HER2-targeted therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4959.
Abstract
It is now well-established that the local microenvironment of an emerging tumor plays a vital role in various steps of tumorigenesis. Stromal cells, such as fibroblasts, endothelial and ...various inflammatory cells, can contribute to tumor progression, and tumor cells can directly model their microenvironment, influencing host cells to secrete factors that favor cancer cell survival and growth. Recent studies have demonstrated the existence of cross-talk between breast cancer (BC) cells and adipocytes, the most abundant stromal cell in the breast, supporting their involvement in BC and consistent with epidemiological studies reporting the association of obesity and insulin resistance/diabetes with a higher risk and poor prognosis of BC.
Based on this evidence, we investigated whether adipocyte de-differentiation is the crucial trigger in inducing pro-tumor microenvironment remodeling and tumor progression.
Co-culture of 4T1 mouse mammary tumor cells using Transwell or their conditioned medium with mature adipocytes derived from the 3T3-L1 mouse cells induced adipocyte de-differentiation, observed as a reduced expression of specific differentiation markers (peripilin, adipocyte protein 2 (aP2), PPARã and adiponectin) in Western blots and by qRT-PCR, and a reduced triglyceride content as assessed by oil red O staining compared to mature adipocytes. Strong adipocyte de-differentiation, associated with an increase in pro-inflammatory molecules (IL6 and CCL2), was also induced in vitro by tumor interstitial fluids obtained from MMT-PyMT transgenic mice, but not by plasma of these mice. Preliminary studies to identify molecules involved in the cross-talk indicated that the phenomenon is exosome-unrelated.
Based on the relevant role of the PPARã transcription factor in inducing and maintaining adipocyte differentiation, we co-cultured adipocytes with 4T1 BC cells in the presence of several PPARã agonists enriched in the diets of many populations displaying a low incidence of cancer (þ3-fatty acids), and in the presence of glitazones (rosiglitazone and pioglitazone) to force adipocyte stable differentiation. While most of these compounds did not block adipocyte de-differentiation induced by tumor cells, pioglitazone and eicosanoid acid showed some activity in inhibiting this de-differentiation in vitro. Treatment of MMTV-PyMT transgenic mice at tumor onset by oral gavage with pioglitazone did not significantly reduce mammary tumor growth compared to controls, whereas eicosanoid acid treatment significantly reduced tumor multiplicity and tumor burden compared to controls.
Overall, our results suggest that tumor-induced adipocyte de-differentiation participates in tumorigenesis and that the blockade of this program can inhibit tumor progression.
Supported by AIRC and Fondazione Cariplo.
Citation Format: Tiziana Triulzi, Valentina Ciravolo, Viola Regondi, Martina Di Modica, Claudia Chiodoni, Elda Tagliabue. Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 748.
The use of trastuzumab as standard treatment for HER2-positive breast carcinomas (BCs) had largely improved the clinical outcome of BCs patients. Unfortunately, many of them do not respond to the ...therapy and the pathological complete response (pCR) is achieved in only 50% of patients even in those with tumours classified as the most sensitive to trastuzumab (i.e. HER2-enriched and high immune infiltration).Given the relevance of the immune system in trastuzumab cytotoxicity and the immune regulatory functions of gut bacteria, this project aims at investigating the role of gut microbiota as extrinsic tumour factor that influences trastuzumab activity.We found that antibiotics abrogated trastuzumab benefit in FVB mice in a microbiota dependent manner. Vancomycin and streptomycin lowered the abundance of Clostridiales bacteria and compromised the recruitment of immune cells in tumour microenvironment. Similar results were obtained in a model of HER2-positive BC spontaneous tumorigenesis, but not in the BALB/c model. In the attempt to improve the response to trastuzumab in FVB mice, the role of lactic acid producing bacteria was explored and an improvement in the antitumor efficacy was observed.Patients treated with neoadjuvant trastuzumab, who achieved pCR (R) were characterized by higher abundance of Clostridiales bacteria and a lower representation of Bacteroidales as compared to those with residual disease (NR) at surgery. Transferring faecal material from R and NR into recipient mice recapitulated the response observed in patients indicating a causal role for commensal bacteria. Furthermore, the unsupervised analysis on patients gut microbiota composition identified two microbiota clusters that significantly discriminated patients according to trastuzumab benefit while no association between microbiota clusters and the HER2-enriched PAM50 molecular classification of tumour biopsies was observed. In conclusion, our data support the role of gut microbiota composition in the therapeutic efficacy of trastuzumab independently of tumour intrinsic characteristics.
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