Comparative Study of the Immune Microenvironment in Heterotopic Tumor Models.

The tumor microenvironment (TME) is pivotal in cancer progression and the response to immunotherapy. A "hot" tumor typically contains immune cells that promote anti-tumor immunity, predicting positive prognosis. "Cold" tumors lack immune cells, suggesting a poor outlook across various cancers. Recent research has focused on converting "cold" tumors into "hot" tumors to enhance the success of immunotherapy. A prerequisite for the studies of the TME is an accurate knowledge of the cell populations of the TME. This study aimed to describe the immune TME of lung and colorectal cancer and melanoma, focusing on lymphoid and myeloid cell populations. We induced heterotopic immunocompetent tumors in C57BL/6 mice, using KP and LLC (Lewis lung carcinoma) cells for lung cancer, MC38 cells for colorectal cancer, and B16-F10 cells for melanoma. Immune cell infiltration was analyzed using multicolor flow cytometry in single-cell suspensions after tumor excision. KP cell tumors showed an abundance of neutrophils and eosinophils; however, they contained much less adaptive immune cells, while LLC cell tumors predominated in monocytes, neutrophils, and monocyte-derived dendritic cells. Monocytes and neutrophils, along with a significant T cell infiltration, were prevalent in MC38 tumors. Lastly, B16-F10 tumors were enriched in macrophages, while showing only moderate T cell presence. In conclusion, our data provide a detailed overview of the immune TME of various heterotopic tumors, highlighting the variabilities in the immune cell profiles of different tumor entities. Our data may be a helpful basis when investigating new immunotherapies, and thus, this report serves as a helpful tool for preclinical immunotherapy research design.

Tumour Infiltrating Lymphocytes (TILs) and immune composition in breast cancer patients from Kenya: Spatial distributions and associations with risk factors and tumour characteristics.

BACKGROUND: The immune landscape of breast cancer (BC) in patients from Sub Saharan Africa is understudied. Our aims were to describe the distribution of Tumour Infiltrating Lymphocytes (TILs) within the intratumoural stroma (sTILs) and the leading/invasive edge stroma (LE-TILs), and to evaluate TILs across BC subtypes with established risk factors and clinical characteristics in Kenyan women. METHODS: Visual quantification of sTILs and LE-TILs were performed on Haematoxylin and eosin -stained pathologically confirmed BC cases based on the International TIL working group guidelines. Tissue Microarrays were constructed and stained with immunohistochemistry (IHC) for CD3, CD4, CD8, CD68, CD20, and FOXP3. Linear and logistic regression models were used to assess associations between risk factors and tumour features with IHC markers and total TILs, after adjusting for other covariates. RESULTS: A total of 226 invasive BC cases were included. Overall, LE-TIL (mean = 27.9, SD = 24.5) proportions were significantly higher than sTIL (mean = 13.5, SD = 15.8). Both sTILs and LE- TILs were predominantly composed of CD3, CD8, and CD68. We found higher TILs to be associated with high KI67/high grade and aggressive tumour subtypes, although these associations varied by TIL locations. Older age at menarche (≥ 15 vs. < 15 years) was associated with higher CD3 (OR: 2.06, 95%CI:1.26-3.37), but only for the intra-tumour stroma. CONCLUSION: The TIL enrichment in more aggressive BCs is similar to previously published data in other populations. The distinct associations of sTIL/LE-TIL measures with most examined factors highlight the importance of spatial TIL evaluations in future studies.

Single-cell chemokine receptor profiles delineate the immune contexture of tertiary lymphoid structures in head and neck squamous cell carcinoma.

Tertiary lymphoid structures (TLSs) are organized aggregates of immune cells associated with favourable prognosis and response to immunotherapy in cancer, but the immune architecture of TLSs remains poorly elucidated. Here, we hypothesize that the spatial architecture of leukocytes in TLSs can be reconstructed de novo, at least partially, by cell-inherent chemokine receptors profiles. Single-cell RNA-sequencing (scRNA-seq) revealed 47 subpopulations of leukocytes in head and neck squamous cell carcinoma (HNSC). Combined with bulk RNA-seq, we observed that CXCR3, CCR7, CCR6, CXCR5, and CCR1 are TLS-associated chemokine receptors. According to the spatial reference, the cellular atlas with TLS-associated chemokine receptors in HNSC TLSs was elaborately portrayed by multiplex immunohistochemistry (mIHC). Subsequently, we explored the functions and evolutionary trajectory of cells distributed in TLSs. Our investigation presents an approach to reconstructing the immune architecture of TLSs, which would help boost the antitumor immune response by inducing neogenesis TLSs in HNSC.

RISING STARS: Heterogeneity and the tumor microenvironment in neuroendocrine prostate cancer.

Neuroendocrine prostate cancer, generally arising late in the disease trajectory, is a heterogeneous subtype that infers a worse prognosis and limited treatment options for patients. Characterization of the complex landscape of this disease subtype and scrutiny of the relationship between tumor cells and cells of the surrounding tumor microenvironment have aided in elucidating some of the mechanisms of neuroendocrine disease biology and have uncovered a multitude of signaling pathways involved in disease transdifferentiation under therapeutic selection. In this review, we discuss current efforts to better understand the heterogeneous landscape of neuroendocrine prostate cancer and summarize research efforts to define the interplay between tumor cells and the microenvironment, with an emphasis on the immune component. Research efforts have uncovered several potential therapeutic approaches that may improve disease outcomes for patients diagnosed with neuroendocrine prostate cancer, including the potential for combination immunotherapies. However, additional research is required to fully address and exploit the contribution of tumor cell and microenvironment heterogeneity in developing effective treatment strategies.

The prognostic impact of the immune signature in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.

Immune Microenvironment Landscape in CNS Tumors and Role in Responses to Immunotherapy.

Despite the important evolution of immunotherapeutic agents, brain tumors remain, in general, refractory to immune therapeutics. Recent discoveries have revealed that the glioma microenvironment includes a wide variety of immune cells in various states that play an important role in the process of tumorigenesis. Anti-tumor immune activity may be occurring or induced in immunogenic hot spots or at the invasive edge of central nervous system (CNS) tumors. Understanding the complex heterogeneity of the immune microenvironment in gliomas will likely be the key to unlocking the full potential of immunotherapeutic strategies. An essential consideration will be the induction of immunological effector responses in the setting of the numerous aspects of immunosuppression and evasion. As such, immune therapeutic combinations are a fundamental objective for clinical studies in gliomas. Through immune profiling conducted on immune competent murine models of glioma and ex vivo human glioma tissue, we will discuss how the frequency, distribution of immune cells within the microenvironment, and immune modulatory processes, may be therapeutically modulated to lead to clinical benefits.

T cells expanded from renal cell carcinoma display tumor-specific CD137 expression but lack significant IFN-γ, TNF-α or IL-2 production.

Metastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies. As only a subset of RCC patients respond, alternative strategies should be explored. Patients refractory to anti-PD-1 therapy may benefit from autologous tumor-infiltrating lymphocyte (TIL) therapy. Even though efficient TIL expansion was reported from RCC lesions, it is not well established how many RCC TIL products are tumor-reactive, how well they produce pro-inflammatory cytokines in response to autologous tumors, and whether their response correlates with the presence of specific immune cells in the tumor lesions. We here compared the immune infiltrate composition of RCC lesions with that of autologous kidney tissue of 18 RCC patients. Tcell infiltrates were increased in the tumor lesions, and CD8+ Tcell infiltrates were primarily of effector memory phenotype. Nine out of 16 (56%) tested TIL products we generated were tumor-reactive, as defined by CD137 upregulation after exposure to autologous tumor digest. Tumor reactivity was found in particular in TIL products originating from tumors with ahigh percentage of infiltrated Tcells compared to autologous kidney, and increased CD25 expression on CD8+ Tcells. Importantly, although TIL products had the capacity to produce the key effector cytokines IFN-γ, TNF-α or IL-2, they failed to produce significant amounts in response to autologous tumor digests. In conclusion, TIL products from RCC lesions contain tumor-reactive Tcells. Their restricted tumor-specific cytokine production requires further investigation of immunosuppressive factors in RCC and subsequent optimization of RCC-derived TIL culture conditions.

The murine Microenvironment Cell Population counter method to estimate abundance of tissue-infiltrating immune and stromal cell populations in murine samples using gene expression.

Quantifying tissue-infiltrating immune and stromal cells provides clinically relevant information for various diseases. While numerous methods can quantify immune or stromal cells in human tissue samples from transcriptomic data, few are available for mouse studies. We introduce murine Microenvironment Cell Population counter (mMCP-counter), a method based on highly specific transcriptomic markers that accurately quantify 16 immune and stromal murine cell populations. We validated mMCP-counter with flow cytometry data and showed that mMCP-counter outperforms existing methods. We showed that mMCP-counter scores are predictive of response to immune checkpoint blockade in cancer mouse models and identify early immune impacts of Alzheimer's disease.

Using epigenetic data to estimate immune composition in admixed samples.

Variation in cell-type decomposition can be of interest to changes in biology by themselves, or a confounder in comparing admixed samples with different phenotypes. Here, I present and review various algorithms that may be used for purposes of deconvolving epigenomic data from admixed samples to yield estimates that may be factored into downstream analyses. More specifically, I present a protocol based on an adaptation of the CIBERSORT algorithm, previously developed for gene-expression, to methylation data.

Statistical Approaches in the Studies Assessing Associations between Human Milk Immune Composition and Allergic Diseases: A Scoping Review.

A growing number of studies are focusing on the associations between human milk (HM) immunological composition and allergic diseases. This scoping review aims to identify statistical methods applied in the field and highlight pitfalls and unmet needs. A comprehensive literature search in MEDLINE and Embase retrieved 13,607 unique records. Following title/abstract screening, 29 studies met the selection criteria and were included in this review. We found that definitions of colostrum and mature milk varied across the studies. A total of 17 out of 29 (59%) studies collected samples longitudinally, but only 12% of these used serial (longitudinal) analyses. Multivariable analysis was used in 45% of the studies, but statistical approaches to modelling varied largely across the studies. Types of variables included as potential confounding factors differed considerably between models. Discrimination analysis was absent from all studies and only a single study reported classification measures. Outcomes of this scoping review highlight lack of standardization, both in data collection and handling, which remains one of the main challenges in the field. Improved standardization could be obtained by a consensus group of researchers and clinicians that could recommend appropriate methods to be applied in future prospective studies, as well as already existing datasets.

Early-Life Compartmentalization of Immune Cells in Human Fetal Tissues Revealed by High-Dimensional Mass Cytometry.

The human fetal immune system must protect the infant against the sudden exposure to a large variety of pathogens upon birth. While it is known that the fetal immune system develops in sequential waves, relatively little is known about the composition of the innate and adaptive immune system in the tissues. Here, we applied high-dimensional mass cytometry to profile the immune system in human fetal liver, spleen, and intestine. With Hierarchical Stochastic Neighbor Embedding (HSNE) we distinguished 177 distinct immune cell clusters, including both previously identified and novel cell clusters. PCA analysis indicated substantial differences between the compositions of the immune system in the different organs. Through dual t-SNE we identified tissue-specific cell clusters, which were found both in the innate and adaptive compartment. To determine the spatial location of tissue-specific subsets we developed a 31-antibody panel to reveal both the immune compartment and surrounding stromal elements through analysis of snap-frozen tissue samples with imaging mass cytometry. Imaging mass cytometry reconstructed the tissue architecture and allowed both the characterization and determination of the location of the various immune cell clusters within the tissue context. Moreover, it further underpinned the distinctness of the immune system in the tissues. Thus, our results provide evidence for early compartmentalization of the adaptive and innate immune compartment in fetal spleen, liver, and intestine. Together, our data provide a unique and comprehensive overview of the composition and organization of the human fetal immune system in several tissues.

Absolute lymphocyte counts at end of induction correlate with distinct immune cell compartments in pediatric B cell precursor acute lymphoblastic leukemia.

Several retrospective studies in children with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) provided clinical evidence that higher absolute lymphocyte counts (ALC) early into treatment significantly correlated with improved relapse-free and overall survival. It still remains unknown, however, whether the predictive role of higher ALCs reflects general bone marrow recovery or a more specific attribute of immune function. To investigate this question, we implemented a prospective observational cohort study in 20 children with BCP ALL on day 29 (D29) of induction chemotherapy and immunophenotyped their lymphoid (T, B and natural killer cells) and myeloid (neutrophils, monocytes, dendritic cells) compartments. In a first evaluation of a cohort treated with Children's Oncology Group-based induction chemotherapy, the immune cell compartments were differentially depleted at D29. Neither gender, risk status, minimal residual disease, nor bone marrow recovery markers correlated with D29 ALC. In contrast, both CD3+ T cell and dendritic cell compartments, which did not correlate with age, significantly correlated with D29 ALC (p < 0.0001). In addition, subset complexity of cellular immune compartments was preserved at D29. This study reveals that D29 ALC significantly correlates with distinct immune cell compartments but not with bone marrow recovery markers, suggesting that higher D29 ALCs may contribute to leukemia control by inducing specific host immune activity.