Purpose
Tumor cells thrive by adapting to the signals in their microenvironment. To adapt, cancer cells activate signaling and transcriptional programs and migrate to establish micro-niches, in ...response to signals from neighboring cells and non-cellular stromal factors. Understanding how the tumor microenvironment evolves during disease progression is crucial to deciphering the mechanisms underlying the functional behavior of cancer cells.
Methods
Multiplex immunohistochemistry, spatial analysis and histological dyes were used to identify and measure immune cell infiltration, cell signal activation and extracellular matrix deposition in low-grade, high-grade astrocytoma and glioblastoma.
Results
We show that lower grade astrocytoma tissue is largely devoid of infiltrating immune cells and extracellular matrix proteins, while high-grade astrocytoma exhibits abundant immune cell infiltration, activation, and extensive tissue remodeling. Spatial analysis shows that most T-cells are restricted to perivascular regions, but bone marrow-derived macrophages penetrate deep into neoplastic cell-rich regions. The tumor microenvironment is characterized by heterogeneous PI3K, MAPK and CREB signaling, with specific signaling profiles correlating with distinct pathological hallmarks, including angiogenesis, tumor cell density and regions where neoplastic cells border the extracellular matrix. Our results also show that tissue remodeling is important in regulating the architecture of the tumor microenvironment during tumor progression.
Conclusion
The tumor microenvironment in malignant astrocytoma, exhibits changes in cell composition, cell signaling activation and extracellular matrix deposition during disease development and that targeting the extracellular matrix, as well as cell signaling activation will be critical to designing personalized therapy.
Objectives
To facilitate disease prognosis and improve precise immunotherapy of gastric cancer (GC) patients, a comprehensive study integrating immune cellular and molecular analyses on tumor tissues ...and peripheral blood was performed.
Methods
The association of GC patients’ outcomes and the immune context of their tumors was explored using multiplex immunohistochemistry (mIHC) and transcriptome profiling. Potential immune dysfunction mechanism/s in the tumors on the systemic level was further examined using mass cytometry (CyTOF) in complementary peripheral blood from selected patients. GC cohorts with mIHC and gene expression profiling data were also used as validation cohorts.
Results
Increased CD4+FOXP3+ T‐cell density in the GC tumor correlated with prolonged survival. Interestingly, CD4+FOXP3+ T cells had a close interaction with CD8+ T cells rather than tumor cells. High densities of CD4+FOXP3+ T cells and CD8+ T cells (High‐High) independently predicted prolonged patient survival. Furthermore, the interferon‐gamma (IFN‐γ) gene signature and PDL1 expression were up‐regulated in this group. Importantly, a subgroup of genomically stable (GS) tumors and tumors with chromosomal instability (CIN) within this High‐High group also had excellent survival. The High‐High GS/CIN tumors were coupled with increased frequencies of Tbet+CD4+ T cells and central memory CD4+ T cells in the peripheral blood.
Conclusion
These novel findings identify the combination of CD8+ T cells and FOXP3+CD4+ T cells as a significant prognostic marker for GC patients, which also could potentially be targeted and applied in the combination therapy with immune checkpoint blockades in precision medicine.
In this work, we show an increased CD4+FOXP3+ T cell density in the tumour core correlated with prolonged survival and CD4+FOXP3+ T cells clustered with CD8+ T cells rather than tumour cells. High density of CD4+FOXP3+ T cells and CD8+ T cells (High‐High) independently predicted prolonged patient survival. These High‐High tumours were coupled with an increased IFN‐γ response, antigen presentation, DCs differentiation and PDL1 upregulation in the local tumours, as well as enrichment of Tbet+ CD4+ T cells and central memory CD4+ T cells circulating in the peripheral blood.
Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using ...multiplex-IHC/immunofluorescence (m-IHC/IF). CD4
or CD8
T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2
γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.
.
Background: Tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T-cell therapies have demonstrated promising, though limited, efficacy against melanoma. Methods: We designed a ...model system to explore the efficacy of dual specific T cells derived from melanoma patient TILs by transduction with a Her2-specific CAR. Results: Metastatic melanoma cells in our biobank constitutively expressed Her2 antigen. CAR-TIL produced greater amounts of IFN compared with parental TIL, when co-cultured with Her2 expressing tumor lines, including autologous melanoma tumor lines, although no consistent increase in cytotoxicity by TIL was afforded by expression of a CAR. Results of an in vivo study in NSG mice demonstrated tumor shrinkage when CAR-TILs were used in an adoptive cell therapy protocol. Conclusion: Potential limitations of transduced TIL in our study included limited proliferative potential and a terminally differentiated phenotype, which would need addressing in further work before consideration of clinical translation.
Strategies aimed at stimulating the immune system against cancer have signaled a new era for designing new effective therapies for patients. Recent breakthroughs in adoptive cellular therapy and in ...using checkpoint inhibitors for some patients have renewed much enthusiasm in this field. However, it has become apparent that tumors can use a multitude of inhibitory networks to effectively reduce antitumor immunity. This review discusses our current knowledge of these immune suppressive mechanisms used by tumors and describes potential new strategies that may counteract this problem resulting in significantly increasing therapeutic outcomes of adoptive immunotherapy in a higher proportion of patients.
Elotuzumab, a humanized monoclonal antibody that binds human signaling lymphocytic activation molecule F7 (hSLAMF7) on myeloma cells, was developed to treat patients with multiple myeloma (MM). ...Elotuzumab has a dual mechanism of action that includes the direct activation of natural killer (NK) cells and the induction of NK cell–mediated antibody-dependent cellular cytotoxicity. This study aimed to characterize the effects of elotuzumab on NK cells in vitro and in patients with MM and to determine whether elotuzumab antitumor activity was improved by programmed death receptor-1 (PD-1) blockade. Elotuzumab promoted NK cell activation when added to a coculture of human NK cells and SLAMF7-expressing myeloma cells. An increased frequency of activated NK cells was observed in bone marrow aspirates from elotuzumab-treated patients. In mouse tumor models expressing hSLAMF7, maximal antitumor efficacy of a murine immunoglobulin G2a version of elotuzumab (elotuzumab-g2a) required both Fcγ receptor–expressing NK cells and CD8+ T cells and was significantly enhanced by coadministration of anti–PD-1 antibody. In these mouse models, elotuzumab-g2a and anti–PD-1 combination treatment promoted tumor-infiltrating NK and CD8+ T-cell activation, as well as increased intratumoral cytokine and chemokine release. These observations support the rationale for clinical investigation of elotuzumab/anti–PD-1 combination therapy in patients with MM.
•The combination of elotuzumab and an anti–PD-1 antibody leads to enhanced antitumor efficacy in mouse models.•Enhanced antitumor activity is likely due to the promotion of tumor-infiltrating NK and T-cell activity.
Objective
The immune system can halt cancer progression by suppressing outgrowth of clinically occult micrometastases in a state of cancer‐immune equilibrium. Cutaneous melanoma provides a unique ...opportunity to study the immune contexture of such lesions, as miniscule skin metastases are accessible to clinical inspection and diagnostic biopsy.
Methods
Here, we analysed by multiplex immunofluorescence microscopy samples from a melanoma patient presenting with an overt and an occult in‐transit metastasis (ITM), the latter of which appeared as a small erythematous papule.
Results
Microarchitecture and immune composition in the two lesions were vastly different. CD4+ and CD8+ T cells accumulated around the margin of the overt SOX10+ Melan A+ ITM but were largely excluded from the tumor centre. By contrast, the occult micrometastasis contained only few SOX10+ Melan A− melanoma cells which were scattered within a dense infiltrate of T cells, including a prominent population of CD103+ CD8+ T cells resembling tissue‐resident memory T (TRM) cells. Notably, almost every single melanoma cell in the micrometastasis was in close proximity to these TRM‐like cells.
Conclusion
Such results support the emerging concept that CD103+ CD8+ TRM cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.
Analysing an occult in‐transit metastasis in a melanoma patient, we identify CD103+CD8+ T cells with a resident memory phenotype as the dominant T‐cell subset. Such results support the emerging concept that CD103+CD8+ tissue‐resident memory T cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.
Abstract
Background: The efficacy of PD-(L)1 inhibitors in patients with trastuzumab-resistant advanced HER2+ breast cancer is poor. Although many HER2 targeted therapies are used clinically, their ...effect on the tumor immune microenvironment (TME) and whether this contributes to efficacy is not understood. Tucatinib is a potent, highly selective, HER2 small molecule tyrosine kinase inhibitor with proven clinical benefit in the advanced setting.
Methods: We used two immunocompetent, HER2+ murine cancer models (trastuzumab-sensitive H2N113 and trastuzumab-resistant fo5) to investigate the effects of tucatinib on tumor growth kinetics, as well as tucatinib anti-tumor efficacy in combination with trastuzumab and PD-1 checkpoint blockade. Effects of tucatinib on the tumor infiltrating lymphocytes were analysed using flow cytometry. To identify genes that were modulated by tucatinib treatment, we performed bulk RNA sequencing on fo5 tumors treated with vehicle or tucatinib. We evaluated healthy donor peripheral blood T cells treated with tucatinib for 96 hours and analysed secreted cytokine levels using cytometric bead array.
Results: Treatment of CD4+ and CD8+ T cells with tucatinib together with TCR stimulation resulted significantly higher levels of IFNγ and TNFα compared with stimulated controls (p < 0.0001), suggesting tucatinib can directly modulate human T cell function. In both murine models, tucatinib significantly inhibited tumor growth in a dose-dependent manner and was observed at doses of 25 mg/kg, 50 mg/kg and 100 mg/kg (p < 0.05 between each dose). Median survival was 16 days in the vehicle group vs 50 days with 100 mg/kg of tucatinib (p < 0.0001). Ex vivo analysis of tumors by flow cytometry showed increased infiltration of NK cells (p=0.01) and, CD8+ T cells with high PD-1 (p = 0.001), TIM-3 (p = 0.009), IFNγ (p = 0.003) and Ki67 (p = 0.0003) expression in Tucatinib treated mice compared to vehicle treated mice. Concomitant with these changes there was a significant reduction in numbers of neutrophils (p = 0.0085) and MHC-II low expressing macrophage populations (p < 0.0001) following tucatininb treatment with an increase in frequency of MHC-II expressing dendritic cells (p < 0.0001) and macrophages (p < 0.0001) suggesting an increase in anti-tumor immunity.
Similarly, in the fo5 model tucatinib treated tumors had significantly higher interferon-γ (IFNγ) produced by CD8+ T cells (p = 0.005). Gene expression profile analysis shows significant enrichment in pathways associated with immune activation, including antigen binding and presentation (p = 0.0002), adaptive immune responses (p = 0.0002) including IFNγ (p = 0.0002) and IFNα (p = 0.0002). In this model, tucatinib in combination with trastuzumab demonstrated significantly better anti-tumor activity (p = 0.03) and survival (p < 0.0001) compared with tucatinib alone, with 33% of mice achieving complete tumour regressions. Tucatinib in combination with PD-1 inhibition also demonstrated significantly greater anti-tumor efficacy compared to tucatinib alone (p = 0.0079) with increased survival (p = 0.05) and 50% of mice achieving complete tumor regression.
Conclusions: This study suggests an anti-tumor immune response may be an important component of the efficacy of tucatinib. This is supported by in vitro data demonstrating tucatinib stimulated human peripheral T cells and in vivo data showing favorable effects on the TME mediated by tucatinib treatment. These changes were associated with improved efficacy when tucatinib was combined with PD-1 inhibition or trastuzumab in the setting of trastuzumab resistance. These findings suggest that the combination of tucatinib and PD-1 inhibition is a rational combination that warrants investigation in the clinical setting, particularly for trastuzumab resistant HER2+ breast tumors.
Citation Format: Ran Li, Sneha Sant, Emmaline Brown, Franco Caramia, Ann Byrne, Kylie Clarke, Michael Neeson, Paul J Neeson, Phillip K Darcy, Scott Peterson, Sherene Loi. Tucatinib favourably modulates the immune microenvironment and synergises with anti-PD1 therapy in a trastuzumab resistant HER2+ murine model abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-04.
Transcriptional dosimetry is an emergent field of radiobiology aimed at developing robust methods for detecting and quantifying absorbed doses using radiation-induced fluctuations in gene expression. ...A combination of RNA sequencing, array-based and quantitative PCR transcriptomics in cellular, murine and various ex vivo human models has led to a comprehensive description of a fundamental set of genes with demonstrable dosimetric qualities. However, these are yet to be validated in human tissue due to the scarcity of in situ-irradiated source material. This represents a major hurdle to the continued development of transcriptional dosimetry. In this study, we present a novel evaluation of a previously reported set of dosimetric genes in human tissue exposed to a large therapeutic dose of radiation. To do this, we evaluated the quantitative changes of a set of dosimetric transcripts consisting of
FDXR, BAX, BCL2, CDKN1A, DDB2, BBC3, GADD45A, GDF15, MDM2, SERPINE1, TNFRSF10B, PLK3, SESN2
and
VWCE
in guided pre- and post-radiation (2 weeks) prostate cancer biopsies from seven patients. We confirmed the prolonged dose-responsivity of most of these transcripts in in situ-irradiated tissue.
BCL2, GDF15
, and to some extent
TNFRSF10B
, were markedly unreliable single markers of radiation exposure. Nevertheless, as a full set, these genes reliably segregated non-irradiated and irradiated tissues and predicted radiation absorption on a patient-specific basis. We also confirmed changes in the translated protein product for a small subset of these dosimeters. This study provides the first confirmatory evidence of an existing dosimetric gene set in less-accessible tissues—ensuring peripheral responses reflect tissue-specific effects. Further work will be required to determine if these changes are conserved in different tissue types, post-radiation times and doses.
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