Definition of a novel breast tumor-specific classifier based on secretome analysis.

BACKGROUND: During cancer development, the normal tissue microenvironment is shaped by tumorigenic events. Inflammatory mediators and immune cells play a key role during this process. However, which molecular features most specifically characterize the malignant tissue remains poorly explored. METHODS: Within our institutional tumor microenvironment global analysis (T-MEGA) program, we set a prospective cohort of 422 untreated breast cancer patients. We established a dedicated pipeline to generate supernatants from tumor and juxta-tumor tissue explants and quantify 55 soluble molecules using Luminex or MSD. Those analytes belonged to five molecular families: chemokines, cytokines, growth factors, metalloproteinases, and adipokines. RESULTS: When looking at tissue specificity, our dataset revealed some breast tumor-specific characteristics, as IL-16, as well as some juxta-tumor-specific secreted molecules, as IL-33. Unsupervised clustering analysis identified groups of molecules that were specific to the breast tumor tissue and displayed a similar secretion behavior. We identified a tumor-specific cluster composed of nine molecules that were secreted fourteen times more in the tumor supernatants than the corresponding juxta-tumor supernatants. This cluster contained, among others, CCL17, CCL22, and CXCL9 and TGF-ß1, 2, and 3. The systematic comparison of tumor and juxta-tumor secretome data allowed us to mathematically formalize a novel breast cancer signature composed of 14 molecules that segregated tumors from juxta-tumors, with a sensitivity of 96.8% and a specificity of 96%. CONCLUSIONS: Our study provides the first breast tumor-specific classifier computed on breast tissue-derived secretome data. Moreover, our T-MEGA cohort dataset is a freely accessible resource to the biomedical community to help advancing scientific knowledge on breast cancer.

The PD-L1/4-1BB Bispecific Antibody-Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner.

PURPOSE: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. EXPERIMENTAL DESIGN: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. RESULTS: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. CONCLUSIONS: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.

Quantitative Systems Pharmacology Approaches for Immuno-Oncology: Adding Virtual Patients to the Development Paradigm.

Drug development in oncology commonly exploits the tools of molecular biology to gain therapeutic benefit through reprograming of cellular responses. In immuno-oncology (IO) the aim is to direct the patient's own immune system to fight cancer. After remarkable successes of antibodies targeting PD1/PD-L1 and CTLA4 receptors in targeted patient populations, the focus of further development has shifted toward combination therapies. However, the current drug-development approach of exploiting a vast number of possible combination targets and dosing regimens has proven to be challenging and is arguably inefficient. In particular, the unprecedented number of clinical trials testing different combinations may no longer be sustainable by the population of available patients. Further development in IO requires a step change in selection and validation of candidate therapies to decrease development attrition rate and limit the number of clinical trials. Quantitative systems pharmacology (QSP) proposes to tackle this challenge through mechanistic modeling and simulation. Compounds' pharmacokinetics, target binding, and mechanisms of action as well as existing knowledge on the underlying tumor and immune system biology are described by quantitative, dynamic models aiming to predict clinical results for novel combinations. Here, we review the current QSP approaches, the legacy of mathematical models available to quantitative clinical pharmacologists describing interaction between tumor and immune system, and the recent development of IO QSP platform models. We argue that QSP and virtual patients can be integrated as a new tool in existing IO drug development approaches to increase the efficiency and effectiveness of the search for novel combination therapies.

Adjustment of dendritic cells to the breast-cancer microenvironment is subset specific.

The functions and transcriptional profiles of dendritic cells (DCs) result from the interplay between ontogeny and tissue imprinting. How tumors shape human DCs is unknown. Here we used RNA-based next-generation sequencing to systematically analyze the transcriptomes of plasmacytoid pre-DCs (pDCs), cell populations enriched for type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s), CD14+ DCs and monocytes-macrophages from human primary luminal breast cancer (LBC) and triple-negative breast cancer (TNBC). By comparing tumor tissue with non-invaded tissue from the same patient, we found that 85% of the genes upregulated in DCs in LBC were specific to each DC subset. However, all DC subsets in TNBC commonly showed enrichment for the interferon pathway, but those in LBC did not. Finally, we defined transcriptional signatures specific for tumor DC subsets with a prognostic effect on their respective breast-cancer subtype. We conclude that the adjustment of DCs to the tumor microenvironment is subset specific and can be used to predict disease outcome. Our work also provides a resource for the identification of potential targets and biomarkers that might improve antitumor therapies.

miR200-regulated CXCL12ß promotes fibroblast heterogeneity and immunosuppression in ovarian cancers.

High-grade serous ovarian cancers (HGSOC) have been subdivided into molecular subtypes. The mesenchymal HGSOC subgroup, defined by stromal-related gene signatures, is invariably associated with poor patient survival. We demonstrate that stroma exerts a key function in mesenchymal HGSOC. We highlight stromal heterogeneity in HGSOC by identifying four subsets of carcinoma-associated fibroblasts (CAF-S1-4). Mesenchymal HGSOC show high content in CAF-S1 fibroblasts, which exhibit immunosuppressive functions by increasing attraction, survival, and differentiation of CD25+FOXP3+ T lymphocytes. The beta isoform of the CXCL12 chemokine (CXCL12ß) specifically accumulates in the immunosuppressive CAF-S1 subset through a miR-141/200a dependent-mechanism. Moreover, CXCL12ß expression in CAF-S1 cells plays a crucial role in CAF-S1 immunosuppressive activity and is a reliable prognosis factor in HGSOC, in contrast to CXCL12α. Thus, our data highlight the differential regulation of the CXCL12α and CXCL12ß isoforms in HGSOC, and reveal a CXCL12ß-associated stromal heterogeneity and immunosuppressive environment in mesenchymal HGSOC.

Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer.

Carcinoma-associated fibroblasts (CAF) are key players in the tumor microenvironment. Here, we characterize four CAF subsets in breast cancer with distinct properties and levels of activation. Two myofibroblastic subsets (CAF-S1, CAF-S4) accumulate differentially in triple-negative breast cancers (TNBC). CAF-S1 fibroblasts promote an immunosuppressive environment through a multi-step mechanism. By secreting CXCL12, CAF-S1 attracts CD4+CD25+ T lymphocytes and retains them by OX40L, PD-L2, and JAM2. Moreover, CAF-S1 increases T lymphocyte survival and promotes their differentiation into CD25HighFOXP3High, through B7H3, CD73, and DPP4. Finally, in contrast to CAF-S4, CAF-S1 enhances the regulatory T cell capacity to inhibit T effector proliferation. These data are consistent with FOXP3+ T lymphocyte accumulation in CAF-S1-enriched TNBC and show how a CAF subset contributes to immunosuppression.

Ligand-receptor dissociated expression explains high TSLP without prognostic impact in human primary head and neck squamous cell carcinoma.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine expressed by epithelial cells during allergic inflammation, and activating dendritic cells (DC). Its expression and functional role in cancer remain controversial. We conducted retrospective (n = 89), and prospective studies including patients with untreated primary head and neck squamous cell carcinoma (HNSCC). We found that TSLP was overexpressed by HNSCC tumor cells, and associated with a highly differentiated status. However, no significant difference in overall and recurrence-free survival was found between patients bearing a tumor with high and low TSLP levels, respectively. Surprisingly, there was no significant association between the levels of TSLP expression, and the number of tumor-infiltrating mature DCLAMP(+) DC. In order to explain the apparent lack of TSLP-induced DC activation, we performed phenotypic and functional experiments on freshly resected tumors. Tumor-infiltrating immune cells, including DC, did not express the TSLP receptor heterodimer (TSLPR chain, IL-7Ralpha chain). Furthermore, freshly sorted blood CD11c(+) DC from healthy donors cultured with tumor-conditioned supernatant exhibited an activated profile, but this was not affected by an anti-TSLP blocking antibody, suggesting a DC activation pathway independent of tumor-derived TSLP. Overall, our results demonstrate that TSLP is overexpressed in HNSCC but its function is hampered by the lack of TSLPR-expressing cells in the tumor microenvironment. Such a dissociated ligand-receptor expression may impact intercellular communication in other immune activation pathways, and tumor types.

No evidence for TSLP pathway activity in human breast cancer.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that primes dendritic cells for Th2 induction. It has been implicated in different types of allergic diseases. Recent work suggested that TSLP could play an important role in the tumor microenvironment and influence tumor progression, in particular in breast cancer. In this study we systematically assessed the production of TSLP at the mRNA and protein levels in several human breast cancer cell lines, large-scale public transcriptomics data sets, and primary human breast tumors. We found that TSLP production was marginal, and concerned less than 10% of the tumors, with very low mRNA and protein levels. In most cases TSLP was undetectable and found to be expressed at lower levels in breast cancer as compared to normal breast tissue. Last, we could not detect any functional TSLP receptor (TSLPR) expression neither on hematopoietic cells nor on stromal cells within the primary tumor microenvironment. We conclude that TSLP-TSLPR pathway activity is not significantly detected within human breast cancer. Taken together, these observations do not support TSLP targeting in breast cancer.

The role of reactive oxygen species and metabolism on cancer cells and their microenvironment.

Compelling evidence show that reactive oxygen species (ROS) levels are finely regulated in the cell and can act as "second messengers" in response to diverse stimuli. In tumor epithelial cells, ROS accumulate abnormally and induce signaling cascades that mediate the oncogenic phenotype. In addition to their impact on tumor epithelial cells, ROS also affect the surrounding cells that constitute the tumor microenvironment. Indeed, ROS production increases tumor angiogenesis, drives the onset of inflammation and promotes conversion of fibroblast into myofibroblasts. These cells, initially identified upon wound healing, exhibit similar properties to those observed in fibroblasts associated with aggressive adenocarcinomas. Indeed, analyses of tumors with distinct severity revealed the existence of multiple distinct co-existing subtypes of carcinoma-associated fibroblasts (CAFs), with specific marker protein profiling. Chronic oxidative stress deeply modifies the proportion of these different fibroblast subtypes, further supporting tumor growth and metastatic dissemination. At last, ROS have been implicated in the metabolic reprogramming of both cancer cells and CAFs, allowing an adaptation to oxidative stress that ultimately promotes tumorigenesis and chemoresistance. In this review, we discuss the role of ROS in cancer cells and CAFs and their impact on tumor initiation, progression, and metastasis.

Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/beta-catenin signaling.

Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible ß-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/ß-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon ß-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/ß-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear ß-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/ß-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/ß-catenin pathway.

Foxp3+ T cells induce perforin-dependent dendritic cell death in tumor-draining lymph nodes.

Regulatory T (Treg) cells limit the onset of effective antitumor immunity, through yet-ill-defined mechanisms. We showed the rejection of established ovalbumin (OVA)-expressing MCA101 tumors required both the adoptive transfer of OVA-specific CD8(+) T cell receptor transgenic T cells (OTI) and the neutralization of Foxp3(+) T cells. In tumor-draining lymph nodes, Foxp3(+) T cell neutralization induced a marked arrest in the migration of OTI T cells, increased numbers of dendritic cells (DCs), and enhanced OTI T cell priming. Using an in vitro cytotoxic assay and two-photon live microscopy after adoptive transfer of DCs, we demonstrated that Foxp3(+) T cells induced the death of DCs in tumor-draining lymph nodes, but not in the absence of tumor. DC death correlated with Foxp3(+) T cell-DC contacts, and it was tumor-antigen and perforin dependent. We conclude that Foxp3(+) T cell-dependent DC death in tumor-draining lymph nodes limits the onset of CD8(+) T cell responses.