Tumor Escape Phenotype in Bladder Cancer Is Associated with Loss of HLA Class I Expression,T-Cell Exclusion and Stromal Changes

Cancer eradication and clinical outcome of immunotherapy depend on tumor cell immunogenicity, including HLA class I (HLA-I) and PD-L1 expression on malignant cells, and on the characteristics of the tumor microenvironment, such as tumor immune infiltration and stromal reaction. Loss of tumor HLA-I is a common mechanism of immune escape from cytotoxic T lymphocytes and is linked to cancer progression and resistance to immunotherapy with the inhibitors of PD-L1/PD-1 signaling. Here we observed that HLA-I loss in bladder tumors is associated with T cell exclusion and tumor encapsulation with stromal elements rich in FAP-positive cells. In addition, PD-L1 upregulation in HLA-I negative tumors demonstrated a correlation with high tumor grade and worse overall- and cancer-specific survival of the patients. These changes define common immuno-morphological signatures compatible with cancer immune escape and acquired resistance to therapeutic interventions across different types of malignancy. They also may contribute to the search of new targets for cancer treatment, such as FAP-expressing cancer-associated fibroblasts, in refractory bladder tumors.     

Changes in Immunogenicity during the Development of Urinary Bladder Cancer: A Preliminary Study

In the present study, we evaluated tumor-infiltrating lymphocytes (TILs) and blood regulatory T lymphocyte (Tregs, CD4+/CD25+/FoxP3+) expression in bladder cancer patients. The number of CD4+, CD8+, CD25+, FoxP3+ and CD20+ TILs was analyzed in association with clinico-pathomorphological features. In more advanced metastasizing tumors, showing non-classic differentiation (ND) and a more aggressive tissue invasion type (TIT), the number of TILs decreased. A low number of CD4+ TILs was associated with poor prognosis. Similarly, Treg frequency before surgery and after surgical treatment was significantly lower in more advanced tumors. The changes in TILs, as well as of local and systemic Tregs, were accompanied by changes in the histological phenotype of urothelial carcinoma regarding pT stage, NDs, TIT, and clinical outcomes. The number of TILs and the frequency of blood Tregs (indicators of antitumor response) may be essential for choosing an immunotherapy that is adjusted to the immune status according to the phase of tumor growth. Moreover, a significant reduction in the number of CD4+ and CD8+ TILs with the development of NDs in more advanced tumors may be associated with lower tumor immunogenicity, resulting in immune tolerance towards tumor tissue. These observations and the tendency of urothelial bladder carcinoma to undergo NDs in a heterogeneous manner during tumor progression suggest complex interactions between bladder cancer immunogenicity and stages of tumor progression.     

Interaction between Fibroblasts and Immune Cells Following DNA Damage Induced by Ionizing Radiation

Cancer-associated fibroblasts (CAF) form the basis of tumor microenvironment and possess immunomodulatory functions by interacting with other cells surrounding tumor, including T lymphocytes, macrophages, dendritic cells and natural killer cells. Ionizing radiation is a broadly-used method in radiotherapy to target tumors. In mammalian cells, ionizing radiation induces various types of DNA damages and DNA damage response. Being unspecific, radiotherapy affects all the cells in tumor microenvironment, including the tumor itself, CAFs and immune cells. CAFs are extremely radio-resistant and do not initiate apoptosis even at high doses of radiation. However, following radiation, CAFs become senescent and produce a distinct combination of immunoregulatory molecules. Radiosensitivity of immune cells varies depending on the cell type due to inefficient DNA repair in, for example, monocytes and granulocytes. In this minireview, we are summarizing recent findings on the interaction between CAF, ionizing radiation and immune cells in the tumor microenvironment.     

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68⁺ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8⁺ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4⁺ and CD8⁺ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8⁺ T cells in HCC.     

Immunoprofiling: An Encouraging Method for Predictive Factors Examination in Lung Cancer Patients Treated with Immunotherapy

The efficiency of immunotherapy using monoclonal antibodies that inhibit immune checkpoints has been proven in many clinical studies and well documented by numerous registration approaches. To date, PD-L1 expression on tumor and immune cells, tumor mutation burden (TMB), and microsatellite instability (MSI) are the only validated predictive factors used for the qualification of cancer patients for immunotherapy. However, they are not the ideal predictive factors. No response to immunotherapy could be observed in patients with high PD-L1 expression, TMB, or MSI. On the other hand, the effectiveness of this treatment method also may occur in patients without PD-L1 expression or with low TMB and with microsatellite stability. When considering the best predictive factor, we should remember that the effectiveness of immunotherapy relies on an overly complex process depending on many factors. To specifically stimulate lymphocytes, not only should their activity in the tumor microenvironment be unlocked, but above all, they should recognize tumor antigens. The proper functioning of the anticancer immune system requires the proper interaction of many elements of the specific and non-specific responses. For these reasons, a multi-parameter analysis of the immune system at its different activity levels is considered a very future-oriented predictive marker. Such complex immunological analysis is performed using modern molecular biology techniques. Based on the gene expression studies, we can determine the content of individual immune cells within the tumor, its stroma, and beyond. This includes all cell types from active memory cytotoxic T cells, M1 macrophages, to exhausted T cells, regulatory T cells, and M2 macrophages. In this article, we summarize the possibilities of using an immune system analysis to predict immunotherapy efficacy in cancer patients. Moreover, we present the advantages and disadvantages of immunoprofiling as well as a proposed future direction for this new method of immune system analysis in cancer patients who receive immunotherapy.     

The Changes of Angiogenesis and Immune Cell Infiltration in the Intra- and Peri-Tumoral Melanoma Microenvironment

Malignant melanoma (MM) urgently needs identification of new markers with better predictive value than currently-used clinical and histological parameters. Cancer cells stimulate the formation of a specialized tumor microenvironment, which reciprocally affects uncontrolled proliferation and migration. However, this microenvironment is heterogeneous with different sub-compartments defined by their access to oxygen and nutrients. This study evaluated microvascular density (MVD), CD3⁺ lymphocytes (TILs) and FOXP3⁺ T-regulatory lymphocytes (Tregs) on formalin-fixed paraffin-embedded tissue sections using light microscopy. We analyzed 82 malignant melanomas, divided according to the AJCC TNM classification into four groups—pT1 (35), pT2 (17), pT3 (18) and pT4 (12)—and 25 benign pigmented nevi. All parameters were measured in both the central areas of tumors (C) and at their periphery (P). A marked increase in all parameters was found in melanomas compared to nevi (p = 0.0001). There was a positive correlation between MVD, TILs, FOXP3⁺ Tregs and the vertical growth phase. The results show that MVD, TILs and FOXP3⁺ Tregs substantially influence cutaneous melanoma microenvironment. We found significant topographic differences of the parameters between central areas of tumors and their boundaries.     

The Adaptive Immune Landscape of the Colorectal Adenoma–Carcinoma Sequence

Background. The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma–carcinoma sequence (ACS). Methods. We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic). Results. The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones. Conclusions. The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.     

The Role of Tumor Microenvironment Cells in Colorectal Cancer (CRC) Cachexia

Cancer cachexia (CC) is a multifactorial syndrome in patients with advanced cancer characterized by weight loss via skeletal-muscle and adipose-tissue atrophy, catabolic activity, and systemic inflammation. CC is correlated with functional impairment, reduced therapeutic responsiveness, and poor prognosis, and is a major cause of death in cancer patients. In colorectal cancer (CRC), cachexia affects around 50–61% of patients, but remains overlooked, understudied, and uncured. The mechanisms driving CC are not fully understood but are related, at least in part, to the local and systemic immune response to the tumor. Accumulating evidence demonstrates a significant role of tumor microenvironment (TME) cells (e.g., macrophages, neutrophils, and fibroblasts) in both cancer progression and tumor-induced cachexia, through the production of multiple procachectic factors. The most important role in CRC-associated cachexia is played by pro-inflammatory cytokines, including the tumor necrosis factor α (TNFα), originally known as cachectin, Interleukin (IL)-1, IL-6, and certain chemokines (e.g., IL-8). Heterogeneous CRC cells themselves also produce numerous cytokines (including chemokines), as well as novel factors called “cachexokines”. The tumor microenvironment (TME) contributes to systemic inflammation and increased oxidative stress and fibrosis. This review summarizes the current knowledge on the role of TME cellular components in CRC-associated cachexia, as well as discusses the potential role of selected mediators secreted by colorectal cancer cells in cooperation with tumor-associated immune and non-immune cells of tumor microenvironment in inducing or potentiating cancer cachexia. This knowledge serves to aid the understanding of the mechanisms of this process, as well as prevent its consequences.     

The Stone Guest: How Does pH Affect Binding Properties of PD-1/PD-L1 Inhibitors?

The interaction between programmed cell death-1 (PD-1) and its ligand PD−L1 activates a coinhibitory signal that blocks T-cell activation, promoting the immune escape process in the tumor microenvironment. Development of monoclonal antibodies targeting and inhibiting PD-1/PD−L1 interaction as anticancer immunotherapies has proved successful in multiple clinical settings and for various types of cancer. Notwithstanding, limitations exist with the use of these biologics, including drug resistance and narrow therapeutic response rate in a majority of patients, that demand for the design of more efficacious small molecule-based immunotherapies. Alteration of pH in the tumor microenvironment is a key factor that is involved in promoting drug resistance, tumor survival and progression. In this study, we have investigated the effect of pH shifts on binding properties of distinct classes of PD−L1 inhibitors, including macrocyclic peptide and small molecules. Results expand structure-activity relationships of PD−L1 inhibitors, providing insights into structural features and physicochemical properties that are useful for the design of ligands that may escape a drug resistance mechanism associated to variable pH conditions of tumor microenvironment.     

The Impact of the Tumor Microenvironment on Macrophage Polarization in Cancer Metastatic Progression

Rather than primary solid tumors, metastasis is one of the hallmarks of most cancer deaths. Metastasis is a multistage event in which cancer cells escape from the primary tumor survive in the circulation and disseminate to distant sites. According to Stephen Paget’s “Seed and Soil” hypothesis, metastatic capacity is determined not only by the internal oncogenic driving force but also by the external environment of tumor cells. Throughout the body, macrophages are required for maintaining tissue homeostasis, even in the tumor milieu. To fulfill these multiple functions, macrophages are polarized from the inflammation status (M1-like) to anti-inflammation status (M2-like) to maintain the balance between inflammation and regeneration. However, tumor cell-enforced tumor-associated macrophages (TAMs) (a high M2/M1 ratio status) are associated with poor prognosis for most solid tumors, such as ovarian cancer. In fact, clinical evidence has verified that TAMs, representing up to 50% of the tumor mass, exert both protumor and immunosuppressive effects in promoting tumor metastasis through secretion of interleukin 10 (IL10), transforming growth factor β (TGFβ), and VEGF, expression of PD-1 and consumption of arginine to inhibit T cell anti-tumor function. However, the underlying molecular mechanisms by which the tumor microenvironment favors reprogramming of macrophages to TAMs to establish a premetastatic niche remain controversial. In this review, we examine the latest investigations of TAMs during tumor development, the microenvironmental factors involved in macrophage polarization, and the mechanisms of TAM-mediated tumor metastasis. We hope to dissect the critical roles of TAMs in tumor metastasis, and the potential applications of TAM-targeted therapeutic strategies in cancer treatment are discussed.     

Atorvastatin Attenuates Programmed Death Ligand-1 (PD-L1) Induction in Human Hepatocellular Carcinoma Cells

Liver cancer is the sixth most common cancer worldwide with high morbidity and mortality. Programmed death ligand 1 (PD-L1) is a major ligand of programmed death 1 receptor (PD1), and PD1/PD-L1 checkpoint acts as a negative regulator of the immune system. Cancers evade the host’s immune defense via PD-L1 expression. This study aimed to investigate the effects of tumor-related cytokines, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) on PD-L1 expression in human hepatocellular carcinoma cells, HepG2. Furthermore, as atorvastatin, a cholesterol-lowering agent, is documented for its immunomodulatory properties, its effect on PD-L1 expression was investigated. In this study, through real-time RT-PCR, Western blot, and immunocytochemistry methods, PD-L1 expression in both mRNA and protein levels was found to be synergistically upregulated in HepG2 by a combination of IFNγ and TNFα, and STAT1 activation was mainly responsible for that synergistic effect. Next, atorvastatin can inhibit the induction of PD-L1 by either IFNγ alone or IFNγ/TNFα combination treatment in HepG2 cells. In conclusion, in HepG2 cells, expression of PD-L1 was augmented by cytokines in the tumor microenvironment, and the effect of atorvastatin on tumor immune response through inhibition of PD-L1 induction should be taken into consideration in cancer patients who have been prescribed atorvastatin.     

Variability in Immunohistochemical Detection of Programmed Death Ligand 1 (PD-L1) in Cancer Tissue Types

In normal cell physiology, programmed death 1 (PD-1) and its ligand, PD-L1, play an immunoregulatory role in T-cell activation, tolerance, and immune-mediated tissue damage. The PD-1/PD-L1 pathway also plays a critical role in immune escape of tumor cells and has been demonstrated to correlate with a poor prognosis of patients with several types of cancer. However, recent reports have revealed that the immunohistochemical (IHC) expression of the PD-L1 in tumor cells is not uniform for the use of different antibodies clones, with variable specificity, often doubtful topographical localization, and with a score not uniquely defined. The purpose of this study was to analyze the IHC expression of PD-L1 on a large series of several human tumors to correctly define its staining in different tumor tissues.     

Oncolytic Virotherapy and Microenvironment in Multiple Myeloma

Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of bone marrow (BM) clonal plasma cells, which are strictly dependent on the microenvironment. Despite the improvement of MM survival with the use of new drugs, MM patients still relapse and become always refractory to the treatment. The development of new therapeutic strategies targeting both tumor and microenvironment cells are necessary. Oncolytic virotherapy represent a promising approach in cancer treatment due to tumor-specific oncolysis and activation of the immune system. Different types of human viruses were checked in preclinical MM models, and the use of several viruses are currently investigated in clinical trials in MM patients. More recently, the use of alternative non-human viruses has been also highlighted in preclinical studies. This strategy could avoid the antiviral immune response of the patients against human viruses due to vaccination or natural infections, which could invalid the efficiency of virotherapy approach. In this review, we explored the effects of the main oncolytic viruses, which act through both direct and indirect mechanisms targeting myeloma and microenvironment cells inducing an anti-MM response. The efficacy of the oncolytic virus-therapy in combination with other anti-MM drugs targeting the microenvironment has been also discussed.     

Anti-PD1/PD-L1 Immunotherapy for Non-Small Cell Lung Cancer with Actionable Oncogenic Driver Mutations

Anti-PD1/PD-L1 immunotherapy has emerged as a standard of care for stage III-IV non-small cell lung cancer (NSCLC) over the past decade. Patient selection is usually based on PD-L1 expression by tumor cells and/or tumor mutational burden. However, mutations in oncogenic drivers such as EGFR, ALK, BRAF, or MET modify the immune tumor microenvironment and may promote anti-PD1/PD-L1 resistance. In this review, we discuss the molecular mechanisms associated with these mutations, which shape the immune tumor microenvironment and may impede anti-PD1/PD-L1 efficacy. We provide an overview of the current clinical data on anti-PD1/PD-L1 efficacy in NSCLC with oncogenic driver mutation.     

WNT16B from Ovarian Fibroblasts Induces Differentiation of Regulatory T Cells through β-Catenin Signal in Dendritic Cells

Treatment for cancer can induce a series of secreted factors into the tumor microenvironment, which can affect cancer progression. Wingless-type MMTV (mouse mammary tumor virus) integration site 16B (WNT16B) is a new member of the WNT family and has been reported to play growth-related roles in previous studies. In this study, we found WNT16B could be expressed and secreted into the microenvironment by human ovarian fibroblasts after DNA damage-associated treatment, including chemotherapy drugs and radiation. We also demonstrated that fibroblast-derived WNT16B could result in accumulation of β-catenin in dendritic cells and secretion of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), which contributed to the differentiation of regulatory T cells in a co-culture environment. These results shed light on the roles of WNT16B in immune regulation, especially in regard to cancer treatment.     

Additive Role of Immune System Infiltration and Angiogenesis in Uveal Melanoma Progression

Uveal melanoma (UM) is a malignant tumor that arises in the melanocytes of the uveal tract. It is the most frequent eye cancer, and despite new therapeutic approaches, prognosis is still poor, with up to 50% of patients developing metastasis with no efficient treatment options available. In contrast to cutaneous melanoma, UM is considered an “immune-cold” tumor due to the low mutational burden and the unique immunosuppressive microenvironment. To gain insight into the role of the UM microenvironment in regard to prognosis and metastatic progression, we have performed a pool analysis characterizing the UM microenvironment by using a bioinformatic approach. A variety of scores based on gene expression measuring stromal infiltration were calculated and used to assess association with prognosis. As a result, the highest immune and stromal scores were associated with poor prognosis. Specifically, stromal cells (fibroblasts and endothelial cells), T cells CD8+, natural killer (NK) cells, and macrophages M1 and M2 infiltration were associated with poor prognosis. Contrary to other tumors, lymphocytic infiltration is related to poor prognosis. Only B cells were associated with more favorable prognosis. UM samples scoring high in both angiogenesis (Angio) and antigen presentation (AP) pathways showed a poor prognosis suggesting an additive role of both functions. Almost all these tumors exhibited a chromosome 3 monosomy. Finally, an enrichment analysis showed that tumors classified as high Angio-high AP also activated metabolic pathways such as glycolysis or PI3K-AKT-MTOR. In summary, our pool analysis identified a cluster of samples with angiogenic and inflammatory phenotypes exhibiting poor prognosis and metabolic activation. Our analysis showed robust results replicated in a pool analysis merging different datasets from different analytic platforms.     

Implication of tumor microenvironment in chemoresistance: tumor-associated stromal cells protect tumor cells from cell death

Tumor development principally occurs following the accumulation of genetic and epigenetic alterations in tumor cells. These changes pave the way for the transformation of chemosensitive cells to chemoresistant ones by influencing the uptake, metabolism, or export of drugs at the cellular level. Numerous reports have revealed the complexity of tumors and their microenvironment with tumor cells located within a heterogeneous population of stromal cells. These stromal cells (fibroblasts, endothelial or mesothelial cells, adipocytes or adipose tissue-derived stromal cells, immune cells and bone marrow-derived stem cells) could be involved in the chemoresistance that is acquired by tumor cells via several mechanisms: (i) cell-cell and cell-matrix interactions influencing the cancer cell sensitivity to apoptosis; (ii) local release of soluble factors promoting survival and tumor growth (crosstalk between stromal and tumor cells); (iii) direct cell-cell interactions with tumor cells (crosstalk or oncologic trogocytosis); (iv) generation of specific niches within the tumor microenvironment that facilitate the acquisition of drug resistance; or (v) conversion of the cancer cells to cancer-initiating cells or cancer stem cells. This review will focus on the implication of each member of the heterogeneous population of stromal cells in conferring resistance to cytotoxins and physiological mediators of cell death.