Fusion Of Tumor Cells Research Articles

Expression of CD68 and CD163 in malignant epithelial cells of oral squamous cell carcinoma: Phenotypic shift or mere cell fusion

Background / purposeThe progression of epithelial to mesenchymal tissue (EMT) is a highly intricate process that facilitates the transformation of cancer cells, allowing them to changeover their characteristic epithelial properties to mesenchymal attributes. This notable change empowers the cells with enhanced mobility and the ability to migrate to distant locations. Furthermore, it is imperative to adopt the idea of macrophage tumor cell fusion to achieve comprehensive considerate of this phenomenon. Our primary objective was to conduct a thorough investigation of macrophage-restricted antigens expression, specifically CD68 and CD163, in malignant epithelial cells of oral cavity squamous cell carcinoma (OSCC) to elucidate aforementioned perceptions. Materials and methodsCD68 and CD163 immunohistochemical expression were assessed in oral squamous cell carcinoma (OSCC), encompassing both the neoplastic cells and the tumor-associated macrophages (TAMs). ResultsBoth CD68 and CD163 antigens were revealed in OSCC malignant epithelial cells in a granular cell pattern, localized in membrane and cytoplasm of tumor cells respectively as well as in the infiltrating TAMs. ConclusionThe macrophage antigens were not limited to the infiltrating tumor-associated macrophages (TAMs), but were also observed in a substantial proportion of OSCC malignant epithelial cells within the tumor parenchyma. This particular expression pattern may represent a subset of tumor cells that have undergone an epithelial to a mesenchymal phenotypic transition. In addition, fusion of macrophages with tumor cells cannot be excluded; both might be associated with increased metastatic activity of OSCC.

Immunotherapeutic allogeneic dendritic cell and autologous tumor cell fusion vaccine alone or combined with radiotherapy in canine oral malignant melanoma is safe and potentially effective.

Immunotherapy represents a promising breakthrough in cancer management and is being explored in canine melanomas. Dendritic cells (DCs) play a crucial role in priming T-cell-mediated immune reactions through the antigen-presenting function. Combining immunotherapy and radiation therapy may generate more substantial anti-cancer efficacy through immunomodulation. Our research reported a preliminary result of the safety and outcome of a kind of immunotherapy, the allogeneic dendritic cell and autologous tumor cell fusion vaccine, alone or in combination with hypofractionated radiation therapy, in canine oral malignant melanoma. Two groups of dogs with histopathological diagnoses of oral malignant melanoma were recruited. In group 1 (DCRT), dogs received a combination of DC fusion vaccine and radiotherapy. In group 2 (DC), dogs received DC fusion vaccine alone. DC vaccination was given once every 2 weeks for four doses. Radiotherapy was performed weekly for five fractions. Dogs that received carboplatin were retrospectively collected as a control group (group 3). Five dogs were included in group 1 (two stage II, three stage III), 11 in group 2 (three stage I/II, eight stage III/IV), and eight (two stage I/II, six stage III/IV) in the control group. Both DC and DCRT were well-tolerated, with only mild adverse events reported, including mucositis, gastrointestinal discomfort, and injection site reactions. The median progression-free intervals in groups 1, 2, and 3 were 214 (95% CI, NA, due to insufficient data), 100 (95% CI, 27-237), and 42 days (95% CI, NA-170), respectively, which were not significantly different. The 1-year survival rates were 20, 54.5, and 12.5% in groups 1, 2, and 3. Dogs in the DCRT group exhibited significantly higher TGF-β signals than the DC group throughout the treatment course, indicating a possible higher degree of immunosuppression. The manuscript demonstrated the safety of dendritic cell/tumor cell fusion vaccine immunotherapy, alone or in combination with radiotherapy. The results support further expansion of this immunotherapy, modification of combination treatment and protocols, and investigation of combining DC vaccine with other treatment modalities. Preclinical Trials, PCTE0000475.

Decoding Metastasis: From Cell Death to Fusion in Cancer Progression.

Metastasis is one of the key concepts in modern oncology, which connects the movement of cancer cells in the body with changes in their characteristics and functions. The review examines the main aspects of metastasis, including theories, facts and discoveries that help to better understand this phenomenon and develop new approaches to its treatment. In this article, we also proposed the theory of cell fusion with the formation of hybrid cells as one of the factors of metastasis. We believe that the fusion of tumor cells with other types of motile cells (leukocytes and bone marrow progenitor cells) may represent an additional mechanism of tumor spread. Cells of bone marrow origin, including cells of the myeloid and macrophage lineages, are the best candidates for heterotypic fusion in regenerative conditions. Events such as cell fusion may play a role in tumor dedifferentiation and progression. We presented a number of arguments and data from our own research that speak in favor of the proposed theory. It should be noted that if the fusion of a normal cell with a tumor cell is one of the possible triggers of tumorigenesis and cancer spread, the mechanisms underlying this process may provide possible new targets for treatment. Therefore, their analysis will expand our arsenal of therapeutic tools by adding completely new targets - cell signaling molecules - and will provide the impetus for reconsidering the tumor microenvironment from a different angle.

Abstract 3693: Molecular and functional characterization of circulating CK+/CD45+ cells in breast cancer

Abstract Background: Circulating tumor cells (CTCs) are considered a key element in metastatic progression and represent a validated prognostic biomarker in breast cancer (BC). CTCs in BC are normally defined as epithelial cells (positive for EPCAM or cytokeratin, CK) lacking the expression of the leukocyte marker CD45. Circulating cells expressing both EPCAM/CK and CD45 (dual positive, DPcells) have however been reported in BC but not investigated. It is believed that DPcells derive from the heterotypic cell fusion of tumor and immune cells, but this hypothesis has not been demonstrated yet. We recently reported, for the first time, the association of DPcells with worse survival in a cohort of 341 BC patients (pts) [Reduzzi et al. ASCO 2022] supporting their tumor origin. Here, we further investigated their malignancy and metastatic potential by molecular profiling and functional experiments. Methods: The study consisted of 2 components: A) Single CTCs and DPcells were enriched from the blood of BC patients with the CellSearch platform, and isolated with the DEPArray system. Single cells underwent whole genome amplification and lowpass whole genome sequencing for single cell copy number alteration (CNA) profiling; B) DPcells and CTCs were investigated in the blood of immunodeficient (NSG) and immunocompetent (FVB) mouse models using various cell lines (LM2, 4T1, MVT1). To test their metastatic potential, DPcells from the primary tumor of FVB donor mice were transplanted into recipient NSG mice, and the metastatic burden in the lungs and liver quantified, in comparison with control CD45- tumor cells. Results: We analyzed 73 DPcells and 18 CTCs collected from 16 BC pts: Aberrant genomes were observed in 28% and 93% of evaluable cells, respectively. When collected from the same pt, CTCs showed clonal CNA profiles, while DPcells were highly heterogeneous. In vivo, DPcells could only be detected in the blood of FVB mice but not in NSG mice. They were very rare, comprising of only about 3% of the total CTC count. When DPcells and control tumor cells were isolated from the primary tumor of 10 donor mice and transplanted into the tail vein of 10 recipient mice, we observed no significant differences in the metastatic colonization of the lungs and liver by the two population, indicating that DPcells do in fact possess metastatic ability, and to a similar extent as control cancer cells. Conclusions: DPcells can have an aberrant genome, supporting their malignancy. Contrary to CTCs, DPcells’ CNA profiles are not clonal and, in vivo, they can only be observed in immunocompetent models; both observations support the tumor/immune fusion hypothesis. Finally, DPcells show metastatic potential in mice, in line with the association with worse survival we previously observed in BC pts. Overall DPcells seem a new CTC subpopulation that should not be neglected anymore. More studies are needed to better understand their origin and phenotypic/molecular features. Citation Format: Carolina Reduzzi, Eleonora Nicolo, Lauren L. Ozimski, Marco Silvestri, Lorenzo Gerratana, Mara S. Serafini, Elisabetta Molteni, Nadia Bayou, Amanda K. Strickland, Huiping Liu, Vera Cappelletti, Nicola Aceto, Massimo Cristofanilli. Molecular and functional characterization of circulating CK+/CD45+ cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3693.

Enhancing targeted cancer therapy through multiple drug delivery by silk peptide nanoparticles

AbstractTargeted therapy drugs exert an essential role in precision therapy in cancer treatments. However, their therapeutic efficiency is greatly limited by the poor water solubility and low bioavailability. Herein, by using an epidermal growth factor receptor (EGFR) targeted drug, erlotinib (ERL), as a representative of targeted therapy drugs, we demonstrate silk peptide (SP) extracted from silkworm cocoons can be used as a drug carrier to co‐load curcumin (CUR) and ERL to achieve dramatically improved antitumor therapeutic efficiency. The dual‐drug loaded nanoparticles (CUR/ERL@SP) with a mean size within 150 nm show significantly enhanced cell internalization, and thus induce more effective inhibition of cancer cell growth. As compared with free drugs, CUR/ERL@SP can more efficiently downregulate the expression of EGFR and other proteins promoting tumor invasion, immunosuppression and cell fusion in EGFR mutant PC‐9 lung cancer cells. The evaluation by using the circulating malignant cells (CMCs) from a lung cancer patient further confirms that CUR/ERL@SP can more effectively inhibit EGFR expression. This study provides a convenient strategy to enhance the therapeutic efficacy of targeted therapy drugs and a facile ex vivo method for evaluating treatment outcomes in personalized therapy.

Abstract P2-26-06: Association between CK+/CD45+ circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) alterations in advanced breast cancer patients

Abstract Background: Circulating tumor cells (CTCs) in breast cancer (BC) are commonly defined as epithelial cells (EPCAM and cytokeratin (CK) positive), lacking the universal blood cell marker CD45. Nonetheless, CTCs expressing both CK and CD45 (= dual-positive, DP cells) can be observed in the blood of cancer patients. Early evidence suggests that DP cells might derive from the fusion of tumor cells and macrophages, and we have previously demonstrated that they present aberrant genomes and are associated with worse prognosis in BC [1,2]. Here, to further investigate the mechanisms/pathways underlying their presence, we analyzed the association between DP cells and circulating tumor DNA (ctDNA) alterations. Methods: Blood samples were collected from patients with advanced BC (aBC), before starting a new line of therapy. All patients were enrolled in a prospective clinical trial. For CTC and DP cells analysis, 7.5 ml of blood collected in CellSave® tubes was processed with the FDA-approved CellSearch® platform (positivity cutoffs were ≥1 cell for DPcells and ≥5 cells for CTCs). For ctDNA analysis, plasma was collected from Streck stabilizing tubes and analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs), insertions/deletions (indels), gene fusions/rearrangements and copy number variations (CNVs), which were then classified into pathways based on previously defined profiles generated on the Cancer Genome Atlas database (RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Associations between ctDNA-detected gene alterations and circulating cell types were analyzed through chi square test, while mutant allele frequency (MAF) and number of detected alterations (NDA) were tested by Mann Whitney test. Results: We analyzed blood samples from 169 patients with luminal-like (n=80), HER2+ (n=34) and triple-negative (n=52) aBC. DPcells were detected in 85 patients (50.3 %, range 0-53), of which 40 (47 %) were CTC-positive and 45 (53%) CTC-negative. Somatic ctDNA alterations were detected in all analyzed samples. In the overall population, the presence of ≥1 DPcell was associated with SNVs in the cell cycle pathway (p = 0.043), a numerically higher incidence was also observed for CNVs in this pathway. SNVs and CNVs in the cell cycle pathway were associated with CTCs ≥ 5 as well (p = 0.005 and p = 0.003, respectively). Moreover, associations with CTCs ≥ 5 were observed for RTK SNVs and CNVs (p = 0.041 and p = 0.046, respectively), PI3K SNVs and CNVs (p = 0.006 and p = 0.007, respectively), MYC SNVs and CNVs (p = 0.042). No associations were observed in terms of MAF and NDA. In the luminal-like subgroup an association was highlighted for CNVs in the cell cycle pathway, p = 0.038. CTCs ≥ 5 were associated with PI3K SNVs (p = 0.031). In the triple-negative subgroup DPcells were associated with SNVs in the RAF pathway (p = 0.041), whereas CTCs ≥ 5 were associated with PI3K SNVs and CNVs (p = 0.044 and p = 0.024, respectively) and RTK SNVs (p = 0.008). In the HER2 positive subgroup, a higher MAF and number of detected SNVs was observed for samples with ≥1 DPcell (p = 0.0286 and p = 0.0099, respectively). Conclusions: The study analyzed ctDNA features associated with canonical and CK+/CD45+ CTCs, showing differential gene alteration profiles. Cell cycle pathway SNVs were common in both CTC populations, while other pathways (RTK, PI3K, MYC and RAF) were significantly altered in a mutually exclusive pattern. These results suggest that DPcells might have a different biological meaning compared to canonical CTCs. More studies need to be conducted to better characterize this understudied CTC subpopulation and understand their specific contribution to cancer progression.

Enhancement of polyethylene glycol-cell fusion efficiency by novel application of transient pressure using a jet injector.

Cell-cell fusion involves the fusion of somatic cells into a single hybrid cell. It is not only a physiological process but also an important cell engineering technology which can be applied to various fields, such as regenerative medicine, antibody engineering, genetic engineering, and cancer therapy. There are three major methods of cell fusion: electrical cell fusion, polyethylene glycol (PEG) cell fusion, and virus-mediated cell fusion. Although PEG cell fusion is the most economical approach and does not require expensive instrumentation, it has a poor fusion rate and induces a high rate of cell cytotoxicity. To improve the fusion rate of the PEG method, we combined it with the pyro-drive jet injector (PJI). PJI provides instant pressure instead of cell agitation to increase the probability of cell-to-cell contact and shorten the distance between cells in the process of cell fusion. Here, we report that this improved fusion method not only decreased cell cytotoxicity during the fusion process, but also increased fusion rate compared with the conventional PEG method. Furthermore, we tested the functionality of cells fused using the PJI-PEG method and found them to be comparable to those fused using the conventional PEG method in terms of their application for dendritic cell (DC)-tumor cell fusion vaccine production; in addition, the PJI-PEG method demonstrated excellent performance in hybridoma cell preparation. Taken together, our data indicate that this method improves cell fusion efficiency as compared to the PEG method and thus has the potential for use in various applications that require cell fusion technology.

Heterogeneity of Circulating Epithelial Cells in Breast Cancer at Single-Cell Resolution: Identifying Tumor and Hybrid Cells.

Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. This study applies single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in nonmetastatic breast cancer patients. CECs are represented by transcriptionally-distinct populations that include both aneuploid and diploid cells. CD45- CECs are predominantly aneuploid, but one population contained more diploid than aneuploid cells. CD45+ CECs mostly diploid: only two populations have aneuploid cells. Diploid CD45+ CECs annotated as different immune cells, surprisingly harbored many copy number aberrations, and positively correlated to tumor grade. It is noteworthy that cancer-associated signaling pathways areabundant only in one aneuploid CD45- CEC population, which may represent an aggressive subset of circulating tumor cells. Thus, CD45- and CD45+ CECs are highly heterogeneous in breast cancer patients and include aneuploid cells, which are most likely circulating tumor and hybrid cells, respectively, and diploid cells. DNA ploidy analysis can be an effective instrument for identifying tumor and hybrid cells among CECs. Further follow-up study is needed to determine which subsets of circulating tumor and hybrid cells contribute to breast cancer metastasis.

CK+/CD45+ (dual-positive) circulating cells are associated with prognosis in patients with advanced breast cancer.

1093 Background: Circulating tumor cells (CTCs) expressing epithelial markers (EPCAM, cytokeratin (CK)) and lacking CD45 (a leukocyte marker) have been associated with poor outcome in many cancer types. Nonetheless, the presence of cells expressing both CK and CD45 (CK+/CD45+), circulating in the blood of cancer patients (pts) have also been reported, but not widely investigated. Early evidence indicates that circulating dual-positive cells (DPcells) are hybrids deriving from the fusion of tumor cells and macrophages. We previously reported that it is possible to detect DPcells in the blood of pts with metastatic breast cancer (BC) and that they are associated with shorter progression-free survival (PFS), in pts with <5 CK+/CD45- CTCs. Here, we investigated the impact of DPcells on overall survival (OS) in pts with advanced BC (aBC). Methods: Blood samples (7.5 ml) were collected from aBC pts before starting a new therapy and processed with the FDA-approved CellSearch platform for CTCs and DPcells enumeration. The prognostic role of CTCs and DPcells was assessed through the Kaplan-Meier method using the log-rank test. Single DPcells were isolated using the DEPArray platform and underwent whole genome amplification and lowpass whole genome sequencing (Ampli1 WGA and Ampli1 Lowpass kits). Results: Blood samples from 341 pts with luminal (n=168), HER2+ (n=76) and triple negative (n=88) BC were analyzed. Of these, 131 samples (38.4%) contained ≥5 CTCs (CTCpos), whereas DPcell were detected in 152 samples (44.6%, range 0-53), of which 66 (43.4%) were CTCpos and 86 (56.6%) CTCneg. Overall, DPcells were associated with a shorter OS: median OS 24.5 vs 35.0 months, p=0.046. However, when analyzing CTCpos and CTCneg separately, only the latter group showed a difference in OS according to DPcells presence. In particular, among CTCneg pts, those with ≥4 DPcells showed a 2.3-fold shorter OS (26.7 vs 60.6 months, p=0.025). Moreover, pts with ≥4 DPcells were less likely to experience a 6-months PFS clinical benefit (p=0.015). Interestingly, in the analysis by BC subtype, DPcells were confirmed to be associated with worse OS only in pts with triple negative BC (median OS 11.5 vs 16.9, p=0.048). To explore the exiology of DPcells, 2 out of 3 cells analyzed after single-cell isolation from 1 patient were confirmed to have copy number alterations (CNA) consistent with malignant cells. CNA and mutational profiling of additional single DPcells and CTCs are ongoing. Conclusions: DPcells are associated with worse OS in aBC pts, with the prognostic impact primarily in pts with <5 CTCs and triple negative BC. This suggests that DPcells might be an alternative way of tumor dissemination in specific pts, in which CK+/CD45- CTCs are less prevalent. More studies are needed to better elucidate DPcell clinical significance in BC, and to confirm their fusion-hybrid origin.

Tumor Hybrid Cells: Nature and Biological Significance.

Metastasis is the leading cause of cancer death and can be realized through the phenomenon of tumor cell fusion. The fusion of tumor cells with other tumor or normal cells leads to the appearance of tumor hybrid cells (THCs) exhibiting novel properties such as increased proliferation and migration, drug resistance, decreased apoptosis rate, and avoiding immune surveillance. Experimental studies showed the association of THCs with a high frequency of cancer metastasis; however, the underlying mechanisms remain unclear. Many other questions also remain to be answered: the role of genetic alterations in tumor cell fusion, the molecular landscape of cells after fusion, the lifetime and fate of different THCs, and the specific markers of THCs, and their correlation with various cancers and clinicopathological parameters. In this review, we discuss the factors and potential mechanisms involved in the occurrence of THCs, the types of THCs, and their role in cancer drug resistance and metastasis, as well as potential therapeutic approaches for the prevention, and targeting of tumor cell fusion. In conclusion, we emphasize the current knowledge gaps in the biology of THCs that should be addressed to develop highly effective therapeutics and strategies for metastasis suppression.

Abstract 586: Expression of CD68 on CKpos/CD45pos circulating cells in breast cancer patients: An additional hint supporting the fusion hybrid theory

Abstract Background: Cells expressing both cytokeratyins (CK) and CD45 (dual-positive, DPcells), circulating in the blood of patients with solid tumors, have been reported for different cancers. Their origin is not clear yet, but one hypothesis is that they are hybrids, deriving from the fusion of tumor cells and macrophages. Recently, we have demonstrated that DPcells can have aberrant genomes, and that, in metastatic breast cancer, they are associated with shorter progression free survival. However, a direct proof of their origin from tumor/macrophage fusion is lacking. Here, we investigated the expression of a macrophage marker (CD68) on DPcells in breast cancer patients. Methods: Blood samples collected from BC patients were analyzed using the CellSearch platform, which allowed for the evaluation of the expression of CK, CD45 and CD68 on circulating cells enriched for EpCAM expression. Based on the marker expression, cells were classified as circulating tumor cells (CTCs, CKpos/CD45neg), and DPcells (CKpos/CD45pos). Results: Twenty-six blood samples were collected from 26 patients with luminal, HER2+, and triple negative BC (n = 13, 5, and 8, respectively). Of these, 6 had non-metastatic (NM) BC (i.e. early breast cancer or locally advanced breast cancer), and 20 had metastatic BC (MBC). Overall, 43 DPcells were identified in 14 (54%) samples (3/6 from NMBC and 11/20 from MBC patients). CD68 expression was detected on 26 DPcells (60%) from 13 samples. In the majority of cases (7/13 samples), CD68pos and CD68neg DPcells were detected together, whereas, in 5 samples, CD68pos DPcells only were present. CD68neg DPcells alone were detected in a single sample. Among the 20 MBC cases, 11 were classified as stage IVaggressive and 9 as stage IVindolent (i.e. with ≥ 5 CTCs and < 5 CTCs, respectively). CD68pos DPcells were present in 5/11 stage IVaggressive and in 5/9 stage IVindolent patients, suggesting that they are independent from CTC-based classification. As expected, CD68 was not detected on the majority of CTCs (796 out of 841), but, interestingly, 5% of CTCs were CD68pos. Conclusions and future perspectives: These preliminary results suggest that DPcells can derive from heterotypic cell fusion between tumor cells and macrophages. This would provide a new perspective on the unique interaction between tumor cells and the microenvironment and the potential role of these new cell entity in promoting disease progression. However, many questions about this subpopulation of circulating cells remain unanswered and future studies are needed to better characterize DPcells at both phenotypic and genomic level, to understand their biological and clinical relevance. Citation Format: Carolina Reduzzi, Paolo D'Amico, Youbin Zhang, Vera Cappelletti, Massimo Cristofanilli. Expression of CD68 on CKpos/CD45pos circulating cells in breast cancer patients: An additional hint supporting the fusion hybrid theory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 586.

4P Immunohybridoma cell-based vaccine for the treatment of patients with castration-resistant prostate cancer

Treatments of various forms of cancer with in vitro prepared dendritic cells of various modalities have been shown to be safe but have not led to the desired efficacy. In this study, we examined the safety and efficacy of an autologous vaccine based on the fusion of patient tumor and dendritic cells (aHyC) in the treatment of chemotherapy-naive patients with castration-resistant prostate cancer (CRPC).

Fusion of Invasive Tumor Cells with Infiltrating Macrophages Fuels Epithelial-Mesenchymal Transition and Adaptive Immune Evasion

Heterotypic interaction between tumor cells and adjacent stromal cells mediates tumor development. However, how tumor heterogeneity commits tumors to the malignant transformation and evasion of immunity against metastasis is poorly understood. Here, we have investigated the fusogenicity of human invasive glioblastoma, triple negative breast cancer and gallbladder cancer cells that are all characterized by mesenchymal cell plasticity. These cells displayed the rigorous ability to fuse with macrophages and augment epithelial-mesenchymal transition (EMT), transforming the fused cells into highly invasive hybrids. YKL-40 (Chitinase-3-like-1), known to promote inflammation and serve as an EMT marker, was essential and sufficient for both cell fusion and the invasiveness of tumor cells that express EMT and tumor-associated macrophage markers. Intriguingly, differential gene profiling of single clones from the hybrids demonstrated that YKL-40 and immune checkpoint protein B7-2 (CD86) were elevated and functioned to independently suppress anti-tumor immune factor levels of CD8 + -cytotoxic T lymphocytes (CTL); thus resulting in escape of immune surveillance. YKL-40 and B7-2 dual shRNA abrogated YKL-40-mediated cell fusion and restored CTL anti-tumor immunity, compromising tumor development in xenografts. Clinically, we found tumor hybrids were present in mesenchymal types of glioblastoma, gallbladder cancer and breast cancer. In addition, YKL-40 expression in glioblastoma was correlated with decreased disease-free survival in patients. Collectively, these data offer novel cellular and molecular mechanisms underpinning immune evasion and tumor malignancy, and suggest a new immunotherapeutic intervention strategy by targeting both YKL-40 and B7-2 in cancer.

B7-1 and GM-CSF enhance the anti-tumor immune effect of DC-tumor fusion vaccine in the treatment of prostate cancer.

The treatment of castration-resistant prostate cancer (CRPC) is always a difficulty in the clinic. Most patients with localized tumor eventually develop CRPC, even if hormone therapy is initially effective. Increasing evidence shows immunotherapy has special advantages compared with traditional therapy in cancer treatment. In this study, we constructed the DC-PC-3 fusion vaccine with B7-1- and GM-CSF-specific modification, and studied its ability to stimulate specific immune response and anti-tumor effect in vitro. The results showed that fusion of DC and tumor cells can improve the expression of associated antigens of DCs. DC-tumor fusion vaccine can strongly promote T cell proliferation and IFN-γ secretion and induce a significant tumor-specific cytotoxic T lymphocyte response. In addition, the B7-1/GM-CSF-modified fusion vaccine showed a more significant anti-tumor effect and greater ability to stimulate the immune response than that without specific modification in vitro. Thus, GM-CSF/B7-1-modified fusion vaccine might be used as a potential therapy strategy for prostate cancer.

Abstract PO-014: Harnessing the heterogeneity of circulating hybrid cells in pancreatic adenocarcinoma

Abstract Pancreatic adenocarcinoma (PDAC) remains one of the most lethal malignancies in the United States and tumor heterogeneity has major implications on the effectiveness of systemic therapies. Comprehensive tumor analysis at the single cell level offers an in-depth examination of neoplastic cells with metastatic potential and supports development of individualized treatment strategies to ultimately improve survival. Hybrid cells are a novel cell population derived from immune-cancer cell fusion within solid tumors and disseminate into peripheral blood, where they can be detected as circulating hybrid cells (CHCs) based on their dual expression of tumor and immune cell epitopes. Compared to conventionally-defined circulating tumor cells (CTCs), CHCs are found in patients at higher levels which allows for more robust phenotypic analyses of tumor cell features and collection for downstream multi-omic profiling. To investigate heterogeneity among disseminated CHCs in patients with PDAC, we assessed for tumor cell features using immunohistochemical analyses of single cells from peripheral blood mononuclear cell smears and by single cell RNA sequencing (scRNA-seq) from FACS-isolated cells. Downstream image analyses was performed Zeiss Zen blue software and scRNA-seq was analyzed using the Seurat analysis package. We determined that PDAC patients harbored heterogeneous populations of CHCs with differential protein expression that mirrored expression in corresponding primary tumors. Further, scRNA-seq analyses identified CHC populations with varying degrees of retained tumor identity, as well as subsets derived from different immune cell lineages. Unsupervised clustering of CHCs with tumor-dominate phenotypes demonstrated high differential expression of cancer related genes with known implications in PDAC tumors and treatment resistant pathways. Inference copy number variation analysis revealed chromosomal amplifications of 1q and 19q along with deletions of 6p, 12q, and 22q, characteristic of PDAC tumors. Additionally, a myriad of KRAS mutations were identified in patient CHCs but absent in normal leukocytes. As the product of tumor and immune cell fusion, CHCs provide a rich source for assessing PDAC heterogeneity through genetic and protein expression analyses. Ultimately, CHCs have great potential as a liquid biopsy in pancreatic cancer which could be harnessed to guide therapy. Citation Format: Brett Scott Walker, Sidharth Sengupta, Michael Parappilly, Aysegul Ors, Jared Fischer, Thomas Sutton, Brett Sheppard, Skye Mayo, Melissa Wong. Harnessing the heterogeneity of circulating hybrid cells in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-014.

Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis.

Hematogenous and lymphogenous cancer metastases are significantly impacted by tumor neovascularization, which predominantly consists of blood vessel-relevant angiogenesis, vasculogenesis, vasculogenic mimicry, and lymphatic vessel-related lymphangiogenesis. Among the endothelial cells that make up the lining of tumor vasculature, a majority of them are tumor-derived endothelial cells (TECs), exhibiting cytogenetic abnormalities of aneuploid chromosomes. Aneuploid TECs are generated from “cancerization of stromal endothelial cells” and “endothelialization of carcinoma cells” in the hypoxic tumor microenvironment. Both processes crucially engage the hypoxia-triggered epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT). Compared to the cancerization process, endothelialization of cancer cells, which comprises the fusion of tumor cells with endothelial cells and transdifferentiation of cancer cells into TECs, is the dominant pathway. Tumor-derived endothelial cells, possessing the dual properties of cancerous malignancy and endothelial vascularization ability, are thus the endothelialized cancer cells. Circulating tumor-derived endothelial cells (CTECs) are TECs shed into the peripheral circulation. Aneuploid CD31+ CTECs, together with their counterpart CD31- circulating tumor cells (CTCs), constitute a unique pair of cellular circulating tumor biomarkers. This review discusses a proposed cascaded framework that focuses on the origins of TECs and CTECs in the hypoxic tumor microenvironment and their clinical implications for tumorigenesis, neovascularization, disease progression, and cancer metastasis. Aneuploid CTECs, harboring hybridized properties of malignancy, vascularization and motility, may serve as a unique target for developing a novel metastasis blockade cancer therapy.

Tumor-Cell-Macrophage Fusion Cells as Liquid Biomarkers and Tumor Enhancers in Cancer.

Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught more attention in recent times. Specifically, fusion of tumor cells with macrophages might lead to the development of metastasis by acquiring features such as genetic and epigenetic heterogeneity, chemotherapeutic resistance, and immune tolerance. In addition to the traditional FDA-approved definition of a CTC (CD45-, EpCAM+, cytokeratins 8+, 18+ or 19+, with a DAPI+ nucleus), an additional circulating cell population has been identified as being potential fusions cells, characterized by distinct, large, polymorphonuclear cancer-associated cells with a dual epithelial and macrophage/myeloid phenotype. Artificial fusion of tumor cells with macrophages leads to migratory, invasive, and metastatic phenotypes. Further studies might investigate whether these have a potential impact on the immune response towards the cancer. In this review, the background, evidence, and potential relevance of tumor cell fusions with macrophages is discussed, along with the potential role of intercellular connections in their formation. Such fusion cells could be a key component in cancer metastasis, and therefore, evolve as a diagnostic and therapeutic target in cancer precision medicine.

The curious phenomenon of dual-positive circulating cells: Longtime overlooked tumor cells.

The presence in the blood of patients with solid tumors of circulating cells expressing both epithelial and leukocyte markers (dual-positive cells, DPcells), has often been reported, though it has never been investigated in detail. A recent study suggested that DPcells are hybrid cells derived from the fusion of tumor cells with macrophages. Such fusion hybrids acquire macrophage-associated features endowing them with accelerated growth, increased motility, enhanced invasion activity and thus, a higher efficiency in metastasis formation. However, no direct evidence proving the tumor origin of circulating DPcells was provided in patients. Here we contribute a review of literature data on DPcells and on the hybrid theory with the aim of putting the current evidence both in a biological and clinical perspective and to generate new hypotheses on the mechanisms underlying tumor progression. To add further biological and clinical context to our literature review, we also report some preliminary data from our laboratory on the identification of DPcells in several solid tumors and confirmation of their malignant genotype, thus classifying them as DP-CTCs.

Tumor Microenvironment and Cell Fusion.

Cell fusion is a highly regulated biological process that occurs under both physiological and pathological conditions. The cellular and extracellular environment is critical for the induction of the cell–cell fusion. Aberrant cell fusion is initiated during tumor progression. Tumor microenvironment is a complex dynamic system formed by the interaction between tumor cells and their surrounding cells. Cell–cell fusion mediates direct interaction between tumor cells and their surrounding cells and is associated with tumor initiation and progression. Various microenvironmental factors affect cell fusion in tumor microenvironment and generate hybrids that acquire genomes of both parental cells and exhibit novel characteristics, such as tumor stem cell-like properties, radioresistance, drug resistance, immune evasion, and enhanced migration and invasion abilities, which are closely related to the initiation, invasion, and metastasis of tumor. The phenotypic characteristics of hybrids are based on the phenotypes of parental cells, and the fusion of tumor cells with diverse types of microenvironmental fusogenic cells is concomitant with phenotypic heterogeneity. This review highlights the types of fusogenic cells in tumor microenvironment that can fuse with tumor cells and their specific significance and summarizes the various microenvironmental factors affecting tumor cell fusion. This review may be used as a reference to develop strategies for future research on tumor cell fusion and the exploration of cell fusion-based antitumor therapies.

An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell destruction for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this study, we explore the efficacy of a conditionally replicating HAdV vector expressing the p14 Fusion-Associated Small Transmembrane (FAST) protein (CRAdFAST) in both immunocompetent and immunodeficient mouse models of cancer. The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect. In the murine 4T1 model of cancer, treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene, but it did not reduce the tumor growth rate in vivo. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion, which led to the formation of large acellular regions within the tumor and significantly reduced the tumor growth rate compared to control vector. Our results indicate that expression of p14 FAST from an oncolytic HAdV can improve vector efficacy for the treatment of cancer.