Primary Tumor Microenvironment Research Articles

A humanized orthotopic tumor microenvironment alters the bone metastatic tropism of prostate cancer cells

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγnull (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context.

Neuroinflammation in Autoimmune Disease and Primary Brain Tumors: The Quest for Striking the Right Balance.

According to classical dogma, the central nervous system (CNS) is defined as an immune privileged space. The basis of this theory was rooted in an incomplete understanding of the CNS microenvironment, however, recent advances such as the identification of resident dendritic cells (DC) in the brain and the presence of CNS lymphatics have deepened our understanding of the neuro-immune axis and revolutionized the field of neuroimmunology. It is now understood that many pathological conditions induce an immune response in the CNS, and that in many ways, the CNS is an immunologically distinct organ. Hyperactivity of neuro-immune axis can lead to primary neuroinflammatory diseases such as multiple sclerosis and antibody-mediated encephalitis, whereas immunosuppressive mechanisms promote the development and survival of primary brain tumors. On the therapeutic front, attempts are being made to target CNS pathologies using various forms of immunotherapy. One of the most actively investigated areas of CNS immunotherapy is for the treatment of glioblastoma (GBM), the most common primary brain tumor in adults. In this review, we provide an up to date overview of the neuro-immune axis in steady state and discuss the mechanisms underlying neuroinflammation in autoimmune neuroinflammatory disease as well as in the development and progression of brain tumors. In addition, we detail the current understanding of the interactions that characterize the primary brain tumor microenvironment and the implications of the neuro-immune axis on the development of successful therapeutic strategies for the treatment of CNS malignancies.

An analysis of the immunological tumor microenvironment of primary tumors and regional lymph nodes in squamous cell lung cancer

BackgroundVarious immune cells that play a central role in antitumor immunity accumulate in primary tumors and regional lymph nodes. Such cellular accumulation and the molecular expression were analyzed to elucidate the immunological tumor microenvironment.MethodsFifty squamous cell lung cancer patients with complete resection were included. Resected specimens from primary lung tumors and regional lymph nodes were immunostained for immune-related molecules, such as CD8, CD103, major histocompatibility complex (MHC) class I, and programmed cell death protein ligand-1 (PD-L1), and the relationship between the prognosis and clinicopathological factors was retrospectively analyzed.ResultsTumor-infiltrating lymphocytes and CD8+ lymphocytes, intratumoral and intrastromal CD103+ lymphocytes, tumor diameter, pathological T and N factors, and pathological stage were significant prognostic factors for the disease-specific survival (DSS) in a univariate analysis. In a multivariate analysis, intratumoral and intrastromal CD103+ lymphocytes and pathological T and N factors were independent prognostic factors of the DSS. Significant concordance was found between the PD-L1 expression of primary tumors and metastatic lymph nodes as well as among tumor-infiltrating lymphocytes, CD8+ lymphocytes and CD103+ lymphocytes. Infiltration of CD103+ lymphocytes into the tumor was significantly correlated with an increased PD-L1 expression of cancer cells in both primary tumors and reginal lymph node metastases. Both the intratumoral infiltration of CD103+ lymphocytes and PD-L1 expression of cancer cells were significantly higher in lymph node metastases than in primary tumors.ConclusionsCD103+ lymphocyte infiltration in the primary tumor was shown to be strongly involved in the prognosis.

Prostate Cancer Dormancy and Reactivation in Bone Marrow.

Prostate cancer has a variable clinical course, ranging from curable local disease to lethal metastatic spread. Eradicating metastatic cells is a unique challenge that is rarely met with the available therapies. Thus, targeting prostate cancer cells in earlier disease states is a crucial window of opportunity. Interestingly, cancer cells migrate from their primary site during pre-cancerous and malignant phases to seed secondary organs. These cells, known as disseminated cancer cells (DCCs), may remain dormant for months or decades before activating to form metastases. Bone marrow, a dormancy-permissive site, is the major organ for housed DCCs and eventual metastases in prostate cancer. The dynamic interplay between DCCs and the primary tumor microenvironment (TME), as well as that between DCCs and the secondary organ niche, controls the conversion between states of dormancy and activation. Here, we discuss recent discoveries that have improved our understanding of dormancy signaling and the role of the TME in modulating the epigenetic reprogramming of DCCs. We offer potential strategies to target DCCs in prostate cancer.

Camrelizumab plus chemotherapy as neoadjuvant therapy for resectable, locally advanced esophageal squamous cell carcinoma (NIC-ESCC2019): A multicenter, open-label, single-arm, phase 2 study.

4028 Background: Despite multidisciplinary therapies, prognosis of pts with resectable esophageal squamous cell carcinoma (ESCC) remains poor. Combining PD-1 blockade to neoadjuvant chemotherapy might be a feasible and effective strategy. Camrelizumab (an anti-PD-1 antibody) was approved for advanced or metastatic ESCC in the second-line setting and showed improved anti-tumor activity and survival benefit when combined with chemotherapy in multiple advanced tumors. Methods: In this NIC-ESCC2019 phase 2 study, histologically or cytologically confirmed ESCC pts (stage II-IVA) were enrolled to receive two cycles of neoadjuvant chemoimmunotherapy (NIC) with camrelizumab (200 mg on day 1) plus nab-paclitaxel (260 mg/m² in total on day 1 and day 8) and cisplatin (75 mg/m² in total on days 1-3) of each 21-day cycle, followed by esophagectomy. The primary endpoint was complete pathologic response (CPR) rate in the primary tumor. Besides, we also explored the relationship between the tumor genomic profile or primary-tumor microenvironment and the pathological response. Results: Between Jan 17, 2020 and Dec 8, 2020, 56 pts were enrolled. 51 pts underwent surgical resection, and all had complete tumor resection. CPR was achieved in 18 (35.3%; 95% CI, 21.7%-48.9%) pts; 12 (23.5%) pts had major pathologic response (MPR), and 21 (41.2%) had incomplete pathological response (IPR). Of note, 16 (31.4%) pts achieved CPR in both primary tumor and lymph nodes. The objective response rate was 66.7% (95% CI, 40.0-70.4). No in-hospital mortality occurred. The most common treatment-related adverse events (TRAEs) were decreased WBC (20 [36%] of 56 pts), vomiting (19 [34%]), and alopecia (18 [32%]). Grade 3 TRAEs only occurred in 6 (11%) pts, and there were no grade 4 or 5 TRAEs. The most common immune-related AEs included grade 1-2 rash maculo-papular (7 [13%]) and reactive cutaneous capillary endothelial proliferation (5 [9%]). Presence of mutations in CREBBP and KMT2D at treatment-naïve time-point was correlated with non-response group (IPR and stable disease) (CREBBP, p = 0.046; KMT2D, p = 0.047). Among the immune populations, CD8+, CD8+PD-1+ and CD8+PD-L1+ T cells increased significantly after two doses of NIC, especially in the CPR+MPR group (CD8+, p = 0.013; CD8+PD-1+, p < 0.001; CD8+PD-L1+, p = 0.068). Conclusions: The addition of camrelizumab to neoadjuvant chemotherapy in ESCC demonstrated promising efficacy with acceptable toxicity, supporting the further investigation in a randomized phase 3 clinical trial. Clinical trial information: NCT04225364. [Table: see text]

Tumor-on-chip modeling of organ-specific cancer and metastasis.

Every year, cancer claims millions of lives around the globe. Unfortunately, model systems that accurately mimic human oncology - a requirement for the development of more effective therapies for these patients - remain elusive. Tumor development is an organ-specific process that involves modification of existing tissue features, recruitment of other cell types, and eventual metastasis to distant organs. Recently, tissue engineered microfluidic devices have emerged as a powerful in vitro tool to model human physiology and pathology with organ-specificity. These organ-on-chip platforms consist of cells cultured in 3D hydrogels and offer precise control over geometry, biological components, and physiochemical properties. Here, we review progress towards organ-specific microfluidic models of the primary and metastatic tumor microenvironments. Despite the field's infancy, these tumor-on-chip models have enabled discoveries about cancer immunobiology and response to therapy. Future work should focus on the development of autologous or multi-organ systems and inclusion of the immune system.

MIF inhibits myeloid derived suppressor cell mediated immunosuppression by promoting an inflammatory M1 phenotypic shift

Cancers of the oral cavity and pharynx contribute 3% of yearly cancer cases in the United States, primarily as head and neck squamous cell carcinoma (HNSCC). HNSCC presents with a five-year survival rate of 66.2%, with primary risk factors being alcohol abuse or human papillomavirus infection. Myeloid derived suppressor cells (MDSC) are a myeloid lineage cell population known to accumulate within HNSCC tumors and promote a pro-tumor microenvironment by suppressing the host immune response against tumor cells. Macrophage migration inhibitory factor (MIF) is a cytokine released by immune cells and mediates inflammatory and chemotactic effects upon interaction with its receptor, CD74, expressed by antigen presenting cells (APC), including MDSC. Given their role in promoting tumor growth, our group sought to elucidate the function of host versus tumor cell derived MIF on MDSC. Given the chemotactic effects of MIF upon APC, we hypothesized that knockdown of Mif gene expression would result in a better outcome and reduced tumor burden as a result of reduced MDSC accumulation within the primary tumor microenvironment. Using murine HNSCC cell lines LY2 and MOC2 in conjunction with Mif+/+, Mif-/- and myeloid specific MIF knockout mice (m-Mif-/-), our group has revealed an anti-immunosuppressive role for host-derived MIF through its interaction with MDSC, with tumor derived MIF being immunosuppressive in nature. After injection of the tumor cell lines into the buccal mucosa of age matched mice, we monitored tumor development until terminal sacrifice. At this point, we extracted tumor cells for flow cytometric analysis and isolated tumor associated Gr-1+ MDSC. Extracted MDSC were co-cultured with naïve T cells fluorescently labeled with CFSE, to enable quantification of suppression of T cell proliferation. Furthermore, we extracted isolated MDSC RNA for analysis by RT-qPCR. Unexpectedly, our initial finding ran contrary to our hypothesis, as we found that Mif-/- mice had a mean tumor burden of 108.3 mg compared to Mif+/+ mice who presented with a mean tumor of only 64 mg (p = 0.00624). Flow cytometry revealed increased CD11b+ F4/80+ macrophages in mice expressing MIF. Interestingly, a smaller proportion of these cells were identified as MDSC in Mif+/+ and m-Mif-/- compared to Mif-/-, determined by expression of MDSC markers Ly6G and Ly6C. Our group found that both Mif+/+ and m-Mif-/- demonstrated reduced suppression of T cell proliferation compared to Mif-/-. Gene expression analysis revealed in Mif-/- MDSC upregulation of immunosuppressive genes Arg1, Ptgs2, S100a8 and S100a9. Seeing that both Mif+/+ and m-Mif-/- demonstrate similar anti-tumor phenotypic and molecular responses to HNSCC, our group has concluded that in response to host derived, but not tumor derived, MIF, MDSC at the tumor site shift towards a more anti-tumor M1 phenotype and additionally that MIF derived from non-myeloid host sources within the tumor microenvironment are sufficient to stimulate this response.

Development of a Novel 3D Model to Investigate the Role of Heterogeneity in Ovarian Cancer Chemoresistance

More than 70% of all patients diagnosed with epithelial ovarian cancer (EOC) succumb to their disease making EOC the most lethal gynecological cancer. The astonishingly high mortality rate of EOC can be explained largely by the fact that 75% of all cases are diagnosed at advanced or metastatic stages thus highlighting the urgency to better understand what cues in the primary tumor microenvironment (TME) promote metastasis. Up to 80% of these patients experience disease reoccurrence demonstrating that in addition to early events in EOC progression, how EOC populations acquire resistance to our current gold standard of treatment is poorly understood. While two-dimensional (2D) in vitro studies of ovarian cancer have provided fundamental insight, three-dimensional (3D) culture systems that more accurately mimic the in vivoTME in terms of biomechanical and biochemical stimuli, are critical research tools in studying ovarian cancer progression. To address this problem, we have developed a 3D culture model that allows for spatial and temporal control of cancer cell interactions with their TME. Our model has the capability to support 3D EOC growth in a mechanically relevant environment over at least a 4-week period while a physically separated fibroblast-rich matrix evolves under the influence of EOC secreted factors. This model maintains the spatial heterogeneity present in both the EOC growth, as well as the developing microenvironment. Using this model, we chose to address two critical gaps in knowledge in the progression of EOC, namely how heterogenous EOC populations acquire resistance and how EOC progression alters surrounding stromal cell populations to promote invasion and metastasis. Our work reveals that altering the dose and administration schedule of paclitaxel can produce heterogenous EOC populations that differ in the acquisition of single or multidrug resistance. By comparing these populations, we found that paclitaxel treatment induced nonuniform metabolic reprogramming across EOC populations which was crucial for the establishment of single and multidrug resistance. Our results provide insight into how current gold standard chemotherapies contribute to the development of chemoresistant EOC subpopulations and describe potential vulnerabilities in these cells that can be exploited for more effective treatment.

Osteoclasts in Tumor Biology: Metastasis and Epithelial-Mesenchymal-Myeloid Transition.

Osteoclast is a specialized cell that originates from monocytic lineage, communicates closely with osteoblasts under physiological conditions, participates in bone modeling and re-modeling, contributes to calcium homeostasis and osteoimmunity. In pathological conditions, it is involved in many tumors such as giant cell bone tumor (osteoclastoma), aneurysmal bone cyst, osteosarcoma, and metastatic cancers, and it usually causes local spread and progression of the tumor, working against the host. Since osteoclasts play an active role in primary bone tumors and bone metastases, the use of anti-osteoclastic agents significantly reduces the mortality and morbidity rates of patients by preventing the progression and local spread of tumors. Osteoclasts also accompany undifferentiated carcinomas of many organs, especially pancreas, thyroid, bladder and ovary. Undifferentiated carcinomas rich in osteoclasts have osteoclastoma-like histology. In these organs, osteoclastoma-like histology may accompany epithelial carcinomas, and de novo, benign and borderline tumors. Mature and immature myeloid cells, including osteoclasts, play an active role in the tumor progression in primary and metastatic tumor microenvironment, in epithelial-mesenchymal transition (EMT), mesenchymal-epithelial-transition (MET), and cancer stem cell formation. Additionally, they are the most suitable candidates for cancer cells in cell fusion due to their evolutionary fusion capabilities. Myeloid features and markers (CD163, CD33, CD68 etc.) can be seen in metastatic cancer cells. Consequently, they provide metastatic cancer cells with motility, margination, transmigration, chemotaxis, phagocytosis, angiogenesis, matrix degradation, and resistance to chemotherapy. For these reasons, we think that the concept of Epithelial-Mesencyhmal-Myeloid-Transition (EMMT) will be more accurate than EMT for cancer cells with myeloid properties.

Tissue-Resident and Recruited Macrophages in Primary Tumor and Metastatic Microenvironments: Potential Targets in Cancer Therapy.

Macrophages within solid tumors and metastatic sites are heterogenous populations with different developmental origins and substantially contribute to tumor progression. A number of tumor-promoting phenotypes associated with both tumor- and metastasis-associated macrophages are similar to innate programs of embryonic-derived tissue-resident macrophages. In contrast to recruited macrophages originating from marrow precursors, tissue-resident macrophages are seeded before birth and function to coordinate tissue remodeling and maintain tissue integrity and homeostasis. Both recruited and tissue-resident macrophage populations contribute to tumor growth and metastasis and are important mediators of resistance to chemotherapy, radiation therapy, and immune checkpoint blockade. Thus, targeting various macrophage populations and their tumor-promoting phenotypes holds therapeutic promise. Here, we discuss various macrophage populations as regulators of tumor progression, immunity, and immunotherapy. We provide an overview of macrophage targeting strategies, including therapeutics designed to induce macrophage depletion, impair recruitment, and induce repolarization. We also provide a perspective on the therapeutic potential for macrophage-specific acquisition of trained immunity as an anti-cancer agent and discuss the therapeutic potential of exploiting macrophages and their traits to reduce tumor burden.

MicroRNA Expression Changes and Integrated Pathways Associated With Poor Outcome in Canine Osteosarcoma.

MicroRNAs (miRNA) are small non-coding RNA molecules involved in post-transcriptional gene regulation. Deregulation of miRNA expression occurs in cancer, and miRNA expression profiles have been associated with diagnosis and prognosis in many cancers. Osteosarcoma (OS), an aggressive primary tumor of bone, affects ~10,000 dogs each year. Though survival has improved with the addition of chemotherapy, up to 80% of canine patients will succumb to metastatic disease. Reliable prognostic markers are lacking for this disease. miRNAs are attractive targets of biomarker discovery efforts due to their increased stability in easily obtained body fluids as well as within fixed tissue. Previous studies in our laboratory demonstrated that dysregulation of genes in aggressive canine OS tumors that participate in miRNA regulatory networks is reportedly disrupted in OS or other cancers. We utilized RT-qPCR in a 384-well-plate system to measure the relative expression of 190 miRNAs in 14 canine tumors from two cohorts of dogs with good or poor outcome (disease-free interval >300 or <100 days, respectively). Differential expression analysis in this subset guided the selection of candidate miRNAs in tumors and serum samples from larger groups of dogs. We ultimately identified a tumor-based three-miR Cox proportional hazards regression model and a serum-based two-miR model, each being able to distinguish patients with good and poor prognosis via Kaplan–Meier analysis with log rank test. Additionally, we integrated miRNA and gene expression data to identify potentially important miRNA–mRNA interactions that are disrupted in canine OS. Integrated analyses of miRNAs in the three-miR predictive model and disrupted genes from previous expression studies suggest the contribution of the primary tumor microenvironment to the metastatic phenotype of aggressive tumors.

Identification of Mitochondrial-Related Prognostic Biomarkers Associated With Primary Bile Acid Biosynthesis and Tumor Microenvironment of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of tumor-associated deaths worldwide. Despite great progress in early diagnosis and multidisciplinary tumor management, the long-term prognosis of HCC remains poor. Currently, metabolic reprogramming during tumor development is widely observed to support rapid growth and proliferation of cancer cells, and several metabolic targets that could be used as cancer biomarkers have been identified. The liver and mitochondria are the two centers of human metabolism at the whole organism and cellular levels, respectively. Thus, identification of prognostic biomarkers based on mitochondrial-related genes (Mito-RGs)—the coding-genes of proteins located in the mitochondria—that reflect metabolic changes associated with HCC could lead to better interventions for HCC patients. In the present study, we used HCC data from The Cancer Genome Atlas (TCGA) database to construct a classifier containing 10 Mito-RGs (ACOT7, ADPRHL2, ATAD3A, BSG, FAM72A, PDK3, PDSS1, RAD51C, TOMM34, and TRMU) for predicting the prognosis of HCC by using 10-fold Least Absolute Shrinkage and Selection Operation (LASSO) cross-validation Cox regression. Based on the risk score calculated by the classifier, the samples were divided into high- and low-risk groups. Gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), t-distributed stochastic neighbor embedding (t-SNE), and consensus clusterPlus algorithms were used to identify metabolic pathways that were significantly different between the high- and low-risk groups. We further investigated the relationship between metabolic status and infiltration of immune cells into HCC tumor samples by using the Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm combined with the Tumor Immune Estimation Resource (TIMER) database. Our results showed that the classifier based on Mito-RGs could act as an independent biomarker for predicting survival of HCC patients. Repression of primary bile acid biosynthesis plays a vital role in the development and poor prognosis of HCC, which provides a potential approach to treatment. Our study revealed cross-talk between bile acid and infiltration of tumors by immune cells, which may provide novel insight into immunotherapy of HCC. Furthermore, our research may provide a novel method for HCC metabolic therapy based on modulation of mitochondrial function.

Role of Exosomes in Prostate Cancer Metastasis.

Prostate cancer remains a life-threatening disease among men worldwide. The majority of PCa-related mortality results from metastatic disease that is characterized by metastasis of prostate tumor cells to various distant organs, such as lung, liver, and bone. Bone metastasis is most common in prostate cancer with osteoblastic and osteolytic lesions. The precise mechanisms underlying PCa metastasis are still being delineated. Intercellular communication is a key feature underlying prostate cancer progression and metastasis. There exists local signaling between prostate cancer cells and cells within the primary tumor microenvironment (TME), in addition to long range signaling wherein tumor cells communicate with sites of future metastases to promote the formation of pre-metastatic niches (PMN) to augment the growth of disseminated tumor cells upon metastasis. Over the last decade, exosomes/ extracellular vesicles have been demonstrated to be involved in such signaling. Exosomes are nanosized extracellular vesicles (EVs), between 30 and 150 nm in thickness, that originate and are released from cells after multivesicular bodies (MVB) fuse with the plasma membrane. These vesicles consist of lipid bilayer membrane enclosing a cargo of biomolecules, including proteins, lipids, RNA, and DNA. Exosomes mediate intercellular communication by transferring their cargo to recipient cells to modulate target cellular functions. In this review, we discuss the contribution of exosomes/extracellular vesicles in prostate cancer progression, in pre-metastatic niche establishment, and in organ-specific metastases. In addition, we briefly discuss the clinical significance of exosomes as biomarkers and therapeutic agents.

Tumor Microenvironment in Metastatic Colorectal Cancer: The Arbitrator in Patients' Outcome.

Simple SummaryColorectal cancer accounts for approximately 10% of all annually diagnosed cancers worldwide being liver metastasis, the most common cause of death in patients with colorectal cancer. The interplay between tumor and stromal cells in the primary tumor microenvironment and at distant metastases are rising in importance as potential mechanisms of the tumor progression. In this review we discuss the new biomarkers derived from tumor microenvironment and liquid biopsy as emerging prognostic and treatments response markers for metastatic colorectal cancer. We also review the developing new clinical strategies based on tumor microenvironmental cells to tackle metastatic disease in metastatic colorectal cancer patients. Colorectal cancer (CRC) is one of the most common cancers in western countries. Its mortality rate varies greatly, depending on the stage of the disease. The main cause of CRC mortality is metastasis, which most commonly affects the liver. The role of tumor microenvironment in tumor initiation, progression and metastasis development has been widely studied. In this review we summarize the role of the tumor microenvironment in the liver pre-metastatic niche formation, paying attention to the distant cellular crosstalk mediated by exosomes. Moreover, and based on the prognostic and predictive capacity of alterations in the stromal compartment of tumors, we describe the role of tumor microenvironment cells and related liquid biopsy biomarkers in the delivery of precise medication for metastatic CRC. Finally, we evaluate the different clinical strategies to prevent and treat liver metastatic disease, based on the targeting of the tumor microenvironment. Specifically, targeting angiogenesis pathways and regulating immune response are two important research pipelines that are being widely developed and promise great benefits.

Abstract PO011: Spatial immune cell and transcriptome profiling uncover differential cellular and molecular signatures in primary and metastatic tumor sites in patients with advanced uterine serous cancer

Abstract Uterine serous carcinoma (USC) is the most aggressive and lethal subtype of endometrial cancer with a low overall patient survival rate of 18%-27%. Extra-uterine metastasis is frequently found in lymph nodes and omentum. The high lethality and poor understanding of the underneath mechanisms that drives USC progression lead to an urgency to develop novel therapeutic tools for the treatment of USC patients. Metastasis is proven to be the leading cause of death from USC and the immune system plays a vital role for this process. However, differential cellular and molecular profiles in the primary and metastatic tumor microenvironment have not been thoroughly examined. To identify the immune cell profile of USC, a panel of 7 immune cell markers together with functional markers granzyme B and Ki-67 were used to examine the spatial localization of a variety of tumor-infiltrating immune cells by multiplex immunofluorescence IHC assay using OpalTM immunology detection kit (Perkin Elmer). Formalin fixed paraffin embedded (FFPE) tissue sections from both primary and metastasis sites (lymph node metastasis) of 10 USC patients were used. Vetra 3.0 microscope was used to take photos of stained slides. Images were quantified and analyzed by inForm software. Transcriptome profiling of microdissected tumor cells from the same tissue samples was performed using the human Clariom D assay platform. Our results demonstrated that primary USC sites had significantly higher density of intratumoral activated tumor-infiltrating cytotoxic T cells (CD8+granzyme B+), and CD8 and LCK double positive T cell subpopulation (CD8+LCK+) than the metastatic sites in lymph nodes. In addition, we found that the density and spatial distribution of activated CD8+ T cells correlate with the overall survival of USC patients. Furthermore, differentially expressed genes and signaling networks associated immune cell activation and tumor cell metastasis were identified in primary sites compared with those in the metastatic sites. Taken together, our study suggested that the immune cell and molecular phenotypes in the lymph nodes provide a permissive niche that support USC progression, which may lead to poor survival rates in patients with advanced USC. Targeting these altered signaling networks may provide novel therapeutic strategies in the treatment of USC patients. Citation Format: Li Zhang, Angela Rynne-Vidal, Karen H. Lu, Samuel C. Mok. Spatial immune cell and transcriptome profiling uncover differential cellular and molecular signatures in primary and metastatic tumor sites in patients with advanced uterine serous cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO011.

Immune Cell Infiltration of the Primary Tumor Microenvironment Predicted the Treatment Outcome of Chemotherapy With or Without Bevacizumab in Metastatic Colorectal Cancer Patients.

BackgroundWith the interest in cancer immunotherapy, it may be possible to combine immunotherapy with bevacizumab and chemotherapy. We evaluated whether tumor-infiltrating immune cells are associated with the efficacy of chemotherapy with or without bevacizumab for the treatment of metastatic colorectal cancer (mCRC).MethodsThis study enrolled mCRC patients on standard treatment with available detailed data and tumor tissue at Sun Yat-sen University Cancer Center between July 1, 2005, and October 1, 2017. CD3+ and CD8+ T cell densities examined by immunohistochemistry in both the tumor core (CT) and invasive margin (IM) were summed as the Immunoscore, and the CD8+/CD3+ T cell ratio was calculated. The predictive and prognostic efficacies of tumor-infiltrating immune cells for progression-free survival (PFS) and overall survival (OS) were analyzed with Kaplan-Meier and Cox analyses.ResultsThe CD8+/CD3+ T cell ratio in the microenvironment was an independent prognostic factor for OS (28.12 mo vs. 16.56 mo, P = 0.017) among the 108 studied patients. In the chemotherapy only group, patients with a high Immunoscore had a high overall response rate (ORR, 40.0% vs. 60.0%, P = 0.022), those with a low CD8+/CD3+ T cell ratio in the microenvironment had a significantly longer PFS (8.64 mo vs. 6.01 mo, P = 0.017), and those with a high CD3+ T cell density in the CT had a longer OS (16.56 mo vs. 25.66 mo, P = 0.029). In the chemotherapy combined with bevacizumab group, patients with a higher CD8+ T cell density in the IM had a longer PFS (7.62 mo vs. 11.66 mo, P = 0.034) and OS (14.55 mo vs. 23.72 mo, P = 0.033).ConclusionImmune cells in primary tumors play an important role in predicting mCRC treatment efficacy. CD8 predicts the effect of bevacizumab plus chemotherapy, while CD3 and CD8/CD3 predict chemotherapy efficacy.

A Highly Reproducible Micro U-Well Array Plate Facilitating High-Throughput Tumor Spheroid Culture and Drug Assessment.

3D multicellular tumor spheroids (MCTSs) have recently emerged as a landmark for cancer research due to their inherent traits that are physiologically relevant to primary tumor microenvironments. A facile approach–laser‐ablated micro U‐wells–has been widely adopted in the past decade. However, the differentiation of microwell uniformities and the construction of arrays have all remained elusive. Herein, an improved laser‐ablated microwell array technique is proposed that can not only achieve arrayed MCTSs with identical sizes but can also perform high‐throughput drug assessments in situ. Three critical laser ablation parameters, including frequency, duty cycle, and pulse number, are investigated to generate microwells flexibly with a range from 170 to 400 μm. The choice of microwells is optimally arranged into an array via precise control of horizontal spacing (d x) and vertical spacing (d y) amenable of cell‐loss‐free culture during cell seeding. Harvested T24, A549 and Huh‐7 MCTSs from the microwell array correspond to approximately 75 to 140 μm in diameter. Anticancer drug screening of cisplatin validated IC50 values in 2D and MCTS conditions are 3.5 versus 9.1 μM (T24), 11.8 versus 277.7 μM (A549) and 33.5 versus 52.8 μM (Huh‐7), and the permeability is measured to range from 0.042 to 0.58 μm min−1.

Clinical Impact of Tumor-Infiltrating Lymphocytes and PD-L1-Positive Cells as Prognostic and Predictive Biomarkers in Urological Malignancies and Retroperitoneal Sarcoma.

Simple SummaryTwo host-dependent biological characteristics, “avoiding immune destruction” and “tumor-promoting inflammation” have been added to cancer hallmarks in 2011. The interaction and cross-talk among tumor cells and several immune cells in a tumor microenvironment are dynamic and complex processes. The purpose of this review is to discuss the prognostic impact of tumor-infiltrating lymphocytes and predictive biomarkers for immune checkpoint inhibitors in four urological solid tumors, the urothelial carcinoma, renal cell carcinoma, prostate cancer, and retroperitoneal sarcoma, through summarizing the findings of observation studies and clinical trials.Over the past decade, an “immunotherapy tsunami”, more specifically that involving immune checkpoint inhibitors (ICIs), has overtaken the oncological field. The interaction and cross-talk among tumor cells and several immune cells in the tumor microenvironment are dynamic and complex processes. As immune contexture can vary widely across different types of primary tumors and tumor microenvironments, there is still a significant lack of clinically available definitive biomarkers to predict patient response to ICIs, especially in urogenital malignancies. An increasing body of evidence evaluating urological malignancies has proven that tumor-infiltrating lymphocytes (TILs) are a double-edged sword in cancer. There is an urgent need to shed light on the functional heterogeneity in the tumor-infiltrating immune system and to explore its prognostic impact following surgery and other treatments. Notably, we emphasized the difference in the immunological profile among urothelial carcinomas arising from different primary origins, the bladder, renal pelvis, and ureter. Significant differences in the density of FOXP3-positive TILs, CD204-positive tumor-infiltrating macrophages, PD-L1-positive cells, and colony-stimulating factors were observed. This review discusses two topics: (i) the prognostic impact of TILs and (ii) predictive biomarkers for ICIs, to shed light on lymphocyte migration in four solid tumors, the urothelial carcinoma, renal cell carcinoma, prostate cancer, and retroperitoneal sarcoma.

Breast Tumor Stiffness Instructs Bone Metastasis Via Mechanical Memory

The mechanical microenvironment of primary breast tumors plays a substantial role in promoting tumor progression1. While the transitory response of cancer cells to pathological stiffness in their native microenvironment has been well described2, it is still unclear how mechanical stimuli in the primary tumor influence distant, late-stage metastatic phenotypes across time and space in absentia. Here, we show that primary tumor stiffness promotes stable, non-genetically heritable phenotypes in breast cancer cells. This mechanical memory instructs cancer cells to adopt and maintain increased cytoskeletal dynamics, traction force, and 3D invasion in vitro, in addition to promoting osteolytic bone metastasis in vivo. Furthermore, we established a mechanical conditioning (MeCo) score comprised of mechanically regulated genes as a global gene expression measurement of tumor stiffness response. Clinically, we show that a high MeCo score is strongly associated with bone metastasis in patients. Using a discovery approach, we mechanistically traced mechanical memory in part to ERK-mediated mechanotransductive activation of RUNX2, an osteogenic gene bookmarker and bone metastasis driver3,4. Expanding our conceptual model, we reveal for the first time that a mechanically sensitive transcription factor can maintain a temporal profile of chromatin accessibility in vitro and in vivo. The combination of these distinctive RUNX2 traits permits the stable transactivation of osteolytic target genes that remain upregulated after cancer cells disseminate from their activating microenvironment in order to modify a distant microenvironment. Using genetic, epigenetic, and functional approaches, we could simulate, repress, select and extend RUNX2-mediated mechanical memory and alter cancer cell behavior accordingly. In concert with previous studies detailing the influence of biochemical properties of the primary tumor stroma on distinct metastatic phenotypes5–9, our findings detailing the influence of biomechanical properties support a generalized model of cancer progression in which the integrated properties of the primary tumor microenvironment govern the secondary tumor microenvironment, i.e., soil instructs soil.

The current state of immunotherapy for primary and secondary brain tumors: similarities and differences.

Treatment and resolution of primary and metastatic brain tumors have long presented a challenge to oncologists. In response to the dismal survival outcomes associated with conventional therapies, various immunotherapy modalities, such as checkpoint inhibitors, vaccine, cellular immunotherapy and viral immunotherapy have been actively explored over the past couple of decades. Although improved patient survival has been more frequently noted in treatment of brain metastases, little progress has been made in improving patient survival in cases of primary brain tumors, specifically glioblastoma, which is the representative primary brain tumor discussed in this review. Herein, we will first overview the findings of recent clinical studies for treatment of primary and metastatic brain tumors with immunotherapeutic interventions. The clinical efficacy of these immunotherapies will be discussed in the context of their ability or inability to overcome inherent characteristics of the tumor as well as restricted antigen presentation and its immunosuppressive microenvironment. Additionally, this review aims to briefly inform clinicians in the field of neuro-oncology on the relevant aspects of the immune system as it pertains to the central nervous system, with special focus on the differing modes of antigen presentation and tumor microenvironment of primary and metastatic brain tumors and the role these differences may play in the efficacy of immunotherapy in eradicating the tumor.