Triple‐negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical ...models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA‐MB‐231‐Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer‐associated adipocyte infiltration in the MDA‐MB‐231‐Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA‐MB‐231‐Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA‐MB‐231‐Luc model. Additionally, we discovered β‐immunoglobulinemia and increased basal levels of G‐CSF correlating with a metastatic switch, with G‐CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread.
What's new?
Triple‐negative breast cancer (TNBC), an aggressive, metastatic, and recurrent breast cancer subtype, currently lacks adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. Here, the authors provide an in‐depth comparative analysis of two preclinical spontaneously metastatic TNBC orthotopic models, MDA‐MB‐231‐Luc and 4T1. The results reveal a metastatic switch in both models with immune system activation and serum‐protein profile reconfiguration, which may support resistance to treatment and recurrence in TNBC. The authors also identify critical functional biomarkers and metabolomic signatures for metastatic progression that may facilitate the development of anticancer therapeutics.
Treatment of triple negative breast cancer (TNBC)-associated metastasis represents an unmet clinical need, and we lack effective therapeutics for a disease that exhibits high relapse rates and ...associates with poor patient outcomes. Advanced nanosized drug delivery systems may enhance the efficacy of first-line chemotherapeutics by altering drug pharmacokinetics and enhancing tumor/metastasis targeting to significantly improve efficacy and safety. Herein, we propose the application of injectable poly-amino acid-based nanogels (NGs) as a versatile hydrophilic drug delivery platform for the treatment of TNBC lung metastasis. We prepared biocompatible and biodegradable cross-linked NGs from polyglutamic acid (PGA) loaded with the chemotherapeutic agent doxorubicin (DOX). Our optimized synthetic procedures generated NGs of ~100 nm in size and 25 wt% drug loading content that became rapidly internalized in TNBC cell lines and displayed IC50 values comparable to the free form of DOX. Importantly, PGA-DOX NGs significantly inhibited lung metastases and almost completely suppressed lymph node metastases in a spontaneously metastatic orthotopic mouse TNBC model. Overall, our newly developed PGA-DOX NGs represent a potentially effective therapeutic strategy for the treatment of TNBC metastases.
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The intrinsic characteristics of the tumor microenvironment (TME), including acidic pH and overexpression of hydrolytic enzymes, offer an exciting opportunity for the rational design of TME-drug ...delivery systems (DDS). We developed and characterized a pH-responsive biodegradable poly-L-glutamic acid (PGA)-based combination conjugate family with the aim of optimizing anticancer effects. We obtained combination conjugates bearing Doxorubicin (Dox) and aminoglutethimide (AGM) with two Dox loadings and two different hydrazone pH-sensitive linkers that promote the specific release of Dox from the polymeric backbone within the TME. Low Dox loading coupled with a short hydrazone linker yielded optimal effects on primary tumor growth, lung metastasis (∼90% reduction), and toxicological profile in a preclinical metastatic triple-negative breast cancer (TNBC) murine model. The use of transcriptomic analysis helped us to identify the molecular mechanisms responsible for such results including a differential immunomodulation and cell death pathways among the conjugates. This data highlights the advantages of targeting the TME, the therapeutic value of polymer-based combination approaches, and the utility of –omics-based analysis to accelerate anticancer DDS.
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Combination nanotherapies for the treatment of breast cancer permits synergistic drug targeting of multiple pathways. However, poor carrier degradability, poor synergism of the combined drugs, low ...drug release regulation, and a lack of control on final macromolecule solution conformation (which drives the biological fate) limit the application of this strategy. The present study describes the development of a family of drug delivery systems composed of chemotherapeutic (doxorubicin) and endocrine therapy (aromatase inhibitor aminoglutethimide) agents conjugated to a biodegradable poly‐l‐glutamic acid backbone via various linking moieties. Data from in vitro cytotoxicity and drug release assessments and animal model validation select a conjugate family member with optimal biological performance. Exhaustive physicochemical characterization in relevant media (including the study of secondary structure, size measurements, and detailed small‐angle neutron scattering analysis) correlates biological data with the intrinsic supramolecular characteristics of the conjugate. Overall, this study demonstrates how a small flexible Gly linker can modify the spatial conformation of the entire polymer–drug conjugate, promote the synergistic release of both drugs, and significantly improve biological activity. These findings highlight the need for a deeper understanding of polymer–drug conjugates at supramolecular level to allow the design of more effective polymer–drug conjugates.
A complex interplay of physical factors drives the interaction between nanomaterials and the biointerface. A panel of physicochemical descriptors allows a structure–anticancer activity relationship to be established for a wide family of biodegradable polyglutamate–drug conjugates, bearing synergistic loadings of chemotherapeutic and endocrine agents, highlighting the crucial role of a small glycine drug linker.
Abstract
PD-1/PD-L1 blockade has greatly improved overall and progression free survival in a variety of cancers. However, only a minority of patients with advanced disease benefit from this approach. ...We have recently showed that inflammasome unleashing by inhibition of the intracellular cation channel TMEM176B with Boritinib (BayK8644) augments anti-PD-1 efficacy. However, the mechanisms involved were not completely understood. We hypothesized that inflammasome activation triggered by Boritinib may recruit Th17 cells to modulate CD8+ T cell exhaustion. To test this hypothesis, we have analyzed Th17 cells and subsets of exhausted CD8+ T cells in the tumor microenvironment of WT mice treated with anti-PD1 ± Boritinib. Combining Boritinib with anti-PD-1 led to an increased frequency of Th17 cells in the tumor microenvironment compared to anti-PD-1 alone. Furthermore, CD73 expression was down-regulated whereas IFN-γ was up-regulated in Th17 cells in the tumor microenvironment of Boritinib + anti-PD-1 treated mice, suggesting an effector phenotype of these cells. In addition, combination of anti-PD-1 with Boritinib results in an increased frequency of progenitor exhausted CD8+ T cells (CD44+PD-1+TCF1+TOX+) and transitory exhausted CD8+ T cells (CD44+PD-1+TCF1−TOX−CX3CR1+) in the tumor microenvironment compared to anti-PD-1 alone. Similar results were obtained using Tmem176b−/− mice treated with anti-PD-1, suggesting an on-target effect of Boritinib. To assess a potential role of Th17 cells in the anti-tumor immunity triggered by Boritinib + anti-PD-1, we treated WT and Il17a−/− mice. We found that Il17 is necessary for the anti-tumor effect of Boritinib + anti-PD-1, since Il17a−/− mice showed a reduced overall survival compared to WT mice. Under this combined therapy, IL-17 may impact on exhausted CD8+ T cells subsets since we found that transitory and terminally exhausted CD8+ T cells in the tumor microenvironment of Il17a−/− mice had less granzyme B than in WT mice. To directly show the role of Th17 cells in the anti-PD-1 anti-tumor immunity we adoptively transferred in vitro differentiated effector Th17 cells. Adoptive transfer of effector Th17 cells into tumor-bearing mice treated with anti-PD-1 resulted in better survival than mice treated with anti-PD-1 alone. To characterize the downstream mechanisms, we performed an in vivo cytotoxicity assay. We found that mice adoptively transferred with effector Th17 cells exhibited greater cytotoxicity by CD8+ T cells than mice treated with anti-PD-1 alone. Furthermore, this could be explained by an increased frequency in progenitor exhausted CD8+ T cells. Thus, effector Th17 cells potentiate CD8+ T cells on anti-PD-1 treatment. In conclusion, combination of anti-PD-1 with Boritinib augments the efficacy of the former by unleashing the inflammasome which results in an increased frequency of Th17 cells leading to modulation of exhausted CD8+ T cells.
Citation Format: Sofia Russo, Mateo Malcuori, David Charbonnier, Mercedes Segovia, Marcelo Hill. Inflammasome unleashing during anti-PD-1 therapy modulates CD8+ T cell exhaustion through Th17 cells. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5116.
Targeting cancer cell exosome release and biogenesis represents a potentially efficient means to treat tumors and prevent cancer recurrence/metastasis; however, the complexity and time-consuming ...nature of currently employed methods to purify and characterize exosomes represent obstacles to progression. Herein, we describe a rapid, convergent, and cost-efficient strategy to analyze candidate U.S. Food and Drug Administration (FDA)-approved drugs that inhibit exosome release and/or biogenesis using breast cancer cell line models in the hope of repurposing them for the clinical treatment of metastatic tumors. We combined the ExoScreen assay based on AlphaScreen
technology with the antibody-mediated detection of an atypical lipid (lysobisphosphatidic acid - LBPA) present in the intra-luminal vesicle/exosomal fraction to achieve both extracellular and intracellular information on exosome modulation after treatment. As proof of concept for this strategy, we identified docetaxel, biscurcumin, primaquine, and doxorubicin as potential exosome release inhibitors in the Her-2 positive MDA-MB-453 and luminal A MCF7 cell lines. Dinaciclib also functioned as an exosome release inhibitor in MCF7 cells. Further, we explored the expression of proteins involved in exosome biogenesis (TSG101, CD9 tetraspanin, Alix, SMase2) and release (Rab11, Rab27) to decipher and validate the possible molecular mechanisms of action of the identified exosome inhibitors. We anticipate that our approach could help to create robust high-throughput screening methodologies to accelerate drug repurposing when using FDA-approved compound libraries and to develop rationally-designed single/combination therapies (including nanomedicines) that can target metastasis progression by modulating exosome biogenesis or release in various tumor types.
Abstract
Our objective is to engineer tumour-targeted polymer-based combination therapies specifically designed to treat metastatic breast cancer (BC) in a personalised manner. Our strategy is to ...develop novel multicomponent polymer conjugates and assess structure activity relationships in clinically relevant models to understand mechanisms of action. Furthermore, we are searching for novel drug combinations, including tumour-derived exosome release pathway inhibitors due to their association with metastasis and tumour drug resistance mechanisms. NCA polymerization techniques have allowed us to precisely control the synthesis of well-defined star-based (STP) and linear (LTP) polypeptidic architectures. After fluorescence labelling, we studied our systems in vitro demonstrating a much more rapid uptake for STP through clathrin-coated mediated endocytosis. pK and biodistribution in healthy mice revealed renal excretion profiles and greater terminal and accumulation half-lives for STP architectures when compared to the LTP. Remarkably, we also observed clear accumulation in immune system-related organs including the spleen and lymph nodes (LN) (up to 40% ID/g tissue accumulation in the LN after i.v. administration). This is possibly due to their inherent structural and morphological features, such as their size (≈100nm), highly negative z-potential values, and/or hydrophilic surfaces. These data highlight the great potential of our stabilized self-assemblies as carriers to target LN metastasis, cancer immunotherapy, or immune system-related approaches, such as vaccination. In parallel, we have performed a High Throughput Screening to select synergistic drug combinations to be used in polymer-based combination approaches through rationally designed linkers that confer adequate drug release kinetics. To perform this approach we selected four metastatic human BC cell lines representing clinical BC subtypes. All cell models have been fully characterized regarding their Cathepsin B activity, intracellular pH, as well as oestrogen, progesterone, Her2 receptors, GSH and exosomes levels; all representing patient-specific biomarkers. Cell viability and exosomes release modulation have been studied following treatments and several drug combinations have been selected for each specific BC subtype. With selected drug combinations different linking chemistry has been designed. We have studied a combination conjugate with the chemotherapeutic agent conjugated to PGA through two different length pH-labile hydrazone linkers. This provided different therapeutic outputs in cells and in a metastatic immunocompetent orthotopic breast cancer model, not only for the primary tumor but also for metastasis progression. The results obtained so far open up a wide range of opportunities for the currently unsuccessful clinical approaches to target LN metastasis and cancer immunotherapy.
Citation Format: Ana Armiñán, Zoraida Andreu, Juan J Arroyo-Crespo, David Charbonnier, Esther Masiá, Fernanda Rodriguez-Otomin, Aroa Duro-Castano, Vicent J Nebot, Maria J Vicent. Design of personalised polymer based combination therapeutics for advanced stage breast cancer patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3726.
Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how ...therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2–related murine β-coronavirus.
Tmem176b
−/−
mice infected with murine β-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical β-coronavirus disease.
Inflammasome-dependent T cell dysfunction is associated with critical β-coronavirus disease and can be rescued by PD-1 blockers.
Regulatory T cells (T
cells) deficient in the transcription factor Foxp3 lack suppressor function and manifest an effector T (T
) cell-like phenotype. We demonstrate that Foxp3 deficiency ...dysregulates metabolic checkpoint kinase mammalian target of rapamycin (mTOR) complex 2 (mTORC2) signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Specific deletion of the mTORC2 adaptor gene Rictor in Foxp3-deficient T
cells ameliorated disease in a Foxo1 transcription factor-dependent manner. Rictor deficiency re-established a subset of T
cell genetic circuits and suppressed the T
cell-like glycolytic and respiratory programs, which contributed to immune dysregulation. Treatment of T
cells from patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their T
cell-like program and restored suppressive function. Thus, regulatory function can be re-established in Foxp3-deficient T
cells by targeting their metabolic pathways, providing opportunities to restore tolerance in T
cell disorders.
The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR ...expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition.
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