Deciphering the tumour immune microenvironment cell by cell.

Immune checkpoint inhibitors (ICIs) have rejuvenated therapeutic approaches in oncology. Although responses tend to be durable, response rates vary in many cancer types. Thus, the identification and validation of predictive biomarkers is a key clinical priority, the answer to which is likely to lie in the tumour microenvironment (TME). A wealth of data demonstrates the huge impact of the TME on ICI response and resistance. However, these data also reveal the complexity of the TME composition including the spatiotemporal interactions between different cell types and their dynamic changes in response to ICIs. Here, we briefly review some of the modalities that sculpt the TME, in particular the metabolic milieu, hypoxia and the role of cancer-associated fibroblasts. We then discuss recent approaches to dissect the TME with a focus on single-cell RNA sequencing, spatial transcriptomics and spatial proteomics. We also discuss some of the clinically relevant findings these multi-modal analyses have yielded.

A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells.

This corrects the article DOI: 10.1038/leu.2016.352.

A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells.

Anti-CD20 monoclonal antibodies (mAb) such as rituximab have been proven to be highly effective at improving outcome in B-cell malignancies. However, many patients ultimately relapse and become refractory to treatment. The glycoengineered anti-CD20 mAb obinutuzumab was developed to induce enhanced antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis and direct cell death and was shown to lead to improved outcomes in a randomized study in B-CLL. We hypothesized that immune stimulation through Toll-like receptor 7 (TLR7) agonism in combination with obinutuzumab would further enhance lymphoma clearance and the generation of long-term antitumor immune responses. Here we demonstrate, in syngeneic human CD20 (hCD20)-expressing models of lymphoma, that systemic administration of a TLR7 agonist (R848) increases responses when administered in combination with obinutuzumab and protects against disease recurrence. Depletion studies demonstrate that primary antitumor activity is dependent on both NK cells and CD4+ T cells but not on CD8+ T cells. However, both CD4+ and CD8+ T cells appear necessary for the generation of protective immunological memory. Importantly, increased tumor-free survival post obinutuzumab and R848 combination therapy was seen in hCD20 transgenic mice, which express hCD20 on normal B cells. These findings provide a rationale for clinical testing of obinutuzumab in combination with systemically administered TLR7 agonists to further improve outcome.

Pre-treatment lymphocytopaenia is an adverse prognostic biomarker in muscle-invasive and advanced bladder cancer.

BACKGROUND: Pre-treatment lymphocytopaenia may result from cytokines secreted by the tumour microenvironment in association with aggressive tumour biology. We sought to establish the prognostic significance of lymphocytopaenia in muscle-invasive and advanced bladder cancer. PATIENTS AND METHODS: Seventy-four patients with muscle-invasive bladder cancer treated with radical chemoradiotherapy and 131 patients with advanced bladder cancer treated with palliative chemotherapy were included in the study. The absolute lymphocyte count on the first day of treatment was recorded. Invasive local or systemic recurrence in the muscle-invasive bladder cancer cohort and all-cause mortality in the advanced bladder cancer cohort were defined as survival end points. Receiver operating characteristic (ROC) curve analysis was utilized to determine the cut-off for defining lymphocytopaenia in the muscle-invasive bladder cancer cohort followed by multivariable analysis in a model evaluating the following variables: anaemia, neutrophilia, tumour stage, hydronephrosis and neoadjuvant chemotherapy. Subsequently, lymphocytopaenia was assessed in a multivariable model of the advanced bladder cancer cohort analysing the following prognostic variables: neutrophilia, anaemia, performance status and presence of bone or visceral metastases. A further analysis was carried out evaluating absolute lymphocyte count as a continuous variable. RESULTS: An absolute lymphocyte count of 1.5 × 10(9)/l was determined as the cut-off on ROC curve analysis in the muscle-invasive bladder cancer cohort, and multivariate analysis revealed that only lymphocytopaenia was predictive for inferior outcome in this cohort. In the advanced bladder cancer cohort, lymphocytopaenia [hazard ratio (HR) 1.6, 95% confidence interval (CI) 1.1-2.4; P = 0.02] and performance status (HR 1.7, 95% CI 1.0-2.7; P = 0.047) were adverse prognostic factors in the binary variable multivariate model. Absolute lymphocyte count was the sole significant factor when analysed as a continuous variable (HR 0.66, 95% CI 0.5-0.87; P = 0.003). CONCLUSION: Pre-treatment lymphocytopaenia is an independent adverse prognostic factor in both muscle-invasive and advanced bladder cancer. It may be a manifestation of cancer-induced immune suppression driving tumour progression.

Stromal infiltration of CD8 T cells is associated with improved clinical outcome in HPV-positive oropharyngeal squamous carcinoma.

BACKGROUND: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a better prognosis than those with HPV-negative tumours. There is interest in de-escalating their treatment but strategies are needed for risk stratification to identify subsets with a poor prognosis. This study investigated tumour-infiltrating lymphocytes (TILs) in relation to HPV tumour status and patient survival. METHODS: Biopsies from 218 patients diagnosed with OPSCC between 2002 and 2011, who underwent chemo/radiotherapy were analysed for HPV by PCR, in-situ hybridisation and p16 immunohistochemistry (IHC). One hundred and thirty-nine samples with concordant HPV detection were analysed for CD3, CD4, CD8 and FoxP3 expression in tumour and stromal regions using multiplexIHC and multispectral image analysis. Labelling of smooth muscle actin (SMA) identified activated stroma. RESULTS: Human papillomavirus-positive compared with HPV-negative OPSCC had higher infiltration in both tumour and stromal areas of CD4 and CD8 T cells but not FoxP3 T regulatory cells. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). There was significantly higher SMA expression in HPV-positive compared with -negative tumours, which did not correlate with survival. CONCLUSIONS: Studies of TILs for risk stratification in OPSCC should assess stromal infiltration.

The antitumor immune response generated by fractionated radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade.

Fractionated radiation therapy (RT) leads to adaptive changes in the tumor microenvironment that may limit the generation of an antitumor immune response. We demonstrated that fractionated RT led to increased tumor cell expression of programmed cell death ligand 1 (PD-L1) in response to CD8+ T cell production of interferon gamma. Our data reveal that the efficacy of fractionated RT can be significantly improved through the generation of durable systemic immune responses when combined with concurrent, but not sequential, blockade of the PD-1/PD-L1 pathway.

Sustained tumour eradication after induced caspase-3 activation and synchronous tumour apoptosis requires an intact host immune response.

Effective anticancer treatments often result in the induction of large amounts of tumour cell death. In vivo, such dying tumour cells are a potential source of antigens for T-cell stimulation. Although apoptosis is generally considered nonimmunogenic, recent evidence suggests that some anticancer therapies that induce apoptosis can elicit antitumour immune responses. Here, a doxycycline-inducible, constitutively active caspase-3 ('death switch') was constructed in a murine tumour model to explore the impact of the host immune response to rapid, synchronous and substantial tumour cell apoptosis. In vitro, up to 80% of tumour cells underwent apoptotic cell death within 24 h and death was accompanied by the release of potential 'danger signal' molecules HMGB1 and HSP90. In vivo, death switch induction provoked rapid, pronounced tumour regression in immune-competent and immune-deficient mice, but sustained tumour eradication was observed only in immune-competent mice. Moreover, the majority of mice that were tumour free after death switch induction were protected from further tumour rechallenge. In addition, long-term remission after induction of the death switch was completely abrogated following depletion of CD8 T cells. These data suggest that sustained tumour eradication after substantial tumour apoptosis requires an antitumour host immune response that prevents tumour relapse. In many patients, cancer therapies produce encouraging initial responses that are only short lived. These results provide new insights that may have important implications for further development of strategies that result in long-term tumour clearance after initially effective anticancer treatment.

Cyclooxygenase-2 inhibition: a potential mechanism for increasing the efficacy of bacillus calmette-guerin immunotherapy for bladder cancer.

PURPOSE: Intravesical bacillus Calmette-Guerin (BCG) therapy is the principal treatment for high risk, noninvasive urothelial carcinoma and carcinoma in situ of the bladder. However, up to 40% of patients fail to respond to this treatment. In this study the potential for inhibition of PGE2 production by BCG treated dendritic cells (DCs) was studied in the context of preferential polarization of the immune response toward a cancer clearing T-helper type 1 immune response. MATERIALS AND METHODS: Murine bone marrow derived DCs were cultured with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. After 7 days the cells were stimulated with BCG. Cell surface expression of co-stimulatory molecules and phagocytic ability were measured by flow cytometry analysis to verify cell activation. The production of IL-10 and IL-12 was measured after DC stimulation with BCG in the presence of IL-10, prostaglandin E2(Cayman Chemical, Ann Arbor, Michigan), antiIL-10 antibody (Insight Biotechnology, Wembley, United Kingdom), NS-398 and indomethacin (Sigma, Poole, United Kingdom). RESULTS: Prostaglandin E2 stimulated a dose dependent increase in the levels of IL-10 produced by BCG activated DCs (p <0.01). IL-10 significantly decreased IL-12 production (p <0.001), while IL-10 blockade significantly increased IL-12 levels (p <0.05). The COX-2 selective inhibitor NS-398 caused a dose dependent increase in the concentration of IL-12 produced by BCG activated DCs (p <0.01). This effect was also seen with the partially selective COX-1 inhibitor indomethacin (p <0.05). CONCLUSIONS: The inhibition of PGE2 synthesis by COX inhibition favored the production of IL-12 by BCG activated DC. This potentially will result in the generation of a T-helper type 1, polarized T-cell response that may improve the efficacy of BCG therapy.