Establishment of patient-derived organotypic tumor spheroid models for tumor microenvironment modeling.

Patient-derived cancer models that reconstitute the characteristics of the tumor microenvironment may facilitate efforts in precision immune-oncology and the discovery of effective anticancer therapies. Organoids that have recently emerged as robust preclinical models typically contain tumor epithelial cells and lack the native tumor immune microenvironment. A patient-derived organotypic tumor spheroid (PDOTS) is a novel and innovative ex vivo system that retains key features of the native tumor immune microenvironment. Here, we established and characterized a series of colorectal cancer PDOTS models for use as a preclinical platform for testing effective immunotherapy and its combinations with other drugs. Partially dissociated (> 100 µm in diameter) tumor tissues were embedded in Matrigel-containing organoid media and subsequently formed into organoid structures within 3 to 7 days of culture. The success rate of growing PDOTS from fresh tissues was ~86%. Morphological analysis showed that the PDOTSs varied in size and structure. Immunofluorescence and flow cytometry analysis revealed that the PDOTSs retained autologous tumor-infiltrating lymphoid cells and tumor-infiltrating lymphoid cells were continually decreased through serial passages. Notably, PDOTSs from tumors from a high-level microsatellite instability-harboring patient were sensitive to anti-PD-1 or anti-PD-L1 antibodies. Our results demonstrate that the PDOTS model in which the tumor immune microenvironment is preserved may represent an advantageous ex vivo system to develop effective immune therapeutics.

Hot or cold: Bioengineering immune contextures into in vitro patient-derived tumor models.

In the past decade, immune checkpoint inhibitors (ICI) have proven to be tremendously effective for a subset of cancer patients. However, it is difficult to predict the response of individual patients and efforts are now directed at understanding the mechanisms of ICI resistance. Current models of patient tumors poorly recapitulate the immune contexture, which describe immune parameters that are associated with patient survival. In this Review, we discuss parameters that influence the induction of different immune contextures found within tumors and how engineering strategies may be leveraged to recapitulate these contextures to develop the next generation of immune-competent patient-derived in vitro models.

In Vivo Antitumor Effect against Murine Cells of CT26 Colon Cancer and EL4 Lymphoma by Autologous Whole Tumor Dead Cells.

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice's immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.

Vaccination with human papillomavirus-18 E1 protein plus α-galactosyl-ceramide induces CD8+ cytotoxic response and impairs the growth of E1-expressing tumors.

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols.

Microfluidic assembly of hydrogel-based immunogenic tumor spheroids for evaluation of anticancer therapies and biomarker release.

Diffuse large B cell lymphoma (DLBCL), the most common subtype of Non-Hodgkin lymphoma, exhibits pathologic heterogeneity and a dynamic immunogenic tumor microenvironment (TME). However, the lack of preclinical in vitro models of DLBCL TME hinders optimal therapeutic screening. This study describes the development of an integrated droplet microfluidics-based platform for high-throughput generation of immunogenic DLBCL spheroids. The spheroids consist of three cell types (cancer, fibroblast and lymphocytes) in a novel hydrogel combination of alginate and puramatrix, which promoted cell adhesion and aggregation. This system facilitates dynamic analysis of cellular interaction, proliferation and therapeutic efficacy via spatiotemporal monitoring and secretome profiling. The immunomodulatory drug lenalidomide had direct anti-proliferative effect on activated B-cell like DLBCL spheroids and reduced several cytokines and other markers (e.g., CCL2, CCL3, CCL4, CD137 and ANG-1 levels) compared with untreated spheroids. Collectively, this novel spheroid platform will enable high-throughput anti-cancer therapeutic screening in a semi-automated manner.

Dendritic-Tumor Fusion Cell-Based Cancer Vaccines.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play a critical role in the induction of antitumor immunity. Therefore, various strategies have been developed to deliver tumor-associated antigens (TAAs) to DCs as cancer vaccines. The fusion of DCs and whole tumor cells to generate DC-tumor fusion cells (DC-tumor FCs) is an alternative strategy to treat cancer patients. The cell fusion method allows DCs to be exposed to the broad array of TAAs originally expressed by whole tumor cells. DCs then process TAAs endogenously and present them through major histocompatibility complex (MHC) class I and II pathways in the context of costimulatory molecules, resulting in simultaneous activation of both CD4⁺ and CD8⁺ T cells. DC-tumor FCs require optimized enhanced immunogenicity of both DCs and whole tumor cells. In this context, an effective fusion strategy also needs to produce immunogenic DC-tumor FCs. We discuss the potential ability of DC-tumor FCs and the recent progress in improving clinical outcomes by DC-tumor FC-based cancer vaccines.

Dendritic Versus Tumor Cell Presentation of Autologous Tumor Antigens for Active Specific Immunotherapy in Metastatic Melanoma: Impact on Long-Term Survival by Extent of Disease at the Time of Treatment.

In patients with metastatic melanoma, sequential single-arm and randomized phase II trials with a therapeutic vaccine consisting of autologous dendritic cells (DCs) loaded with antigens from self-renewing, proliferating, irradiated autologous tumor cells (DC-TC) showed superior survival compared with similar patients immunized with irradiated tumor cells (TC). We wished to determine whether this difference was evident in cohorts who at the time of treatment had (1) no evidence of disease (NED) or (2) had detectable disease. Eligibility criteria and treatment schedules were the same for all three trials. Pooled data confirmed that overall survival (OS) was longer in 72 patients treated with DC-TC compared with 71 patients treated with TC (median OS 60 versus 22 months; 5-year OS 51% versus 32%, p=0.004). Treatment with DC-TC was associated with longer OS in both cohorts. Among 70 patients who were NED at the time that treatment was started, OS was better for DC-TC: 5-year OS 73% versus 43% (p=0.015). Among 73 patients who had detectable metastases, OS was better for DC-TC: median 38.8 months versus 14.7 months, 5-year OS 33% versus 20% (p=0.025). This approach is promising as an adjunct to other therapies in patients who have had metastatic melanoma.

[Therapeutic efficacy of dendritic cells pulsed by autologous tumor cell lysate in combination with CIK cells on advanced renal cell carcinoma].

OBJECTIVE: To investigate the clinical efficacy of dendritic cells (DCs) sensitization by autologous tumor cell lysate in combination with cytokine-induced killer (CIK) cells on patients with advanced renal cell carcinoma and detect their immune functions and adverse effects. METHODS: The study analyzed retrospectively 82 patients with advanced renal cell carcinoma admitted in our department from January 2011 to December 2013. Peripheral blood mononuclear cells (PBMCs) were isolated from the above patients. Adherent cells were cultured to produce DCs. The DCs were pulsed with autologous tumor cell lysate (Ag) to produce Ag-DC. T lymphocytes were cultured to prepare CIK. The Ag-DC was co-cultured with CIK to produce Ag-DC-CIK vaccine, and then the phenotypes of the DCs and the secretion of IL-12 were evaluated. CIK cell proliferation was determined, too. The 41 patients received the immunotherapy of Ag-DC-CIK, and the other 41 patients received CIK cell therapy alone. After 2 cycles of treatment, the changes of T cell subtypes and cytokines released in the peripheral blood were evaluated. The therapeutic outcomes were evaluated with the help of imageology. The adverse effects were also observed. RESULTS: Compared with the DCs alone, DCs pulsed with autologous tumor cell lysates expressed significantly surface molecules CD11c, CD83, CD86 and HLA-DR, and the release of IL-12 significantly increased. CIK cell proliferation was significantly enhanced after co-cultured with the Ag-DC. Meanwhile, the percentages of CD3⁺CD8⁺ cells and CD3⁺CD56⁺ cells went up significantly compared with the controls. Clinical responses of the Ag-DC-CIK treatment group were much better than those of the control group. No severe adverse effects were seen in the two groups. CONCLUSION: DCs derived from PBMCs of advanced renal cell carcinoma patients harboring the autologous tumor cell antigens can differentiate into mature DCs, which facilitate the proliferation of CIK cells. Ag-DC-CIK vaccine improves the immune status of patients with advanced renal cell carcinoma.

Significantly increased CD70 up regulation on TEL-AML positive B cell precursor acute lymphoblastic leukemia cells following CD40 stimulation.

BACKGROUND: TEL-AML the most common genetic alteration in childhood precursor B acute lymphoblastic leukemia (BCP-ALL) is associated with a favorable prognosis. PATIENTS AND METHOD: We studied the expression of nerve growth factor/tumor necrosis factor receptor (NGFR/TNFR)/ligand family members on 108 primary BCP-ALL samples by flow cytometry and compared both their baseline expression and CD40-induced modulation on TEL-AML positive and negative leukemia samples. RESULTS: Our findings demonstrate that TEL-AML positive patients exhibit a significantly higher percentage of CD40, CD27 and p75NTR positive blasts at diagnosis. This might well contribute to the improved relapse-free survival of these patients assessed in Kaplan Meier analysis as CD27 and p75NTR directly mediate apoptotic signals. Furthermore CD40 ligation enhances antigen presenting and T cell stimulatory capacity via significant up regulation of CD70 while adequate response to physiological maturation signals as indicated by concomitant down regulation of CD27 is retained in TEL-AML positive leukemia. CONCLUSION: These data provide novel insights in immunological control mechanisms preserved in this leukemia subtype and suggest that not only treatment with chemicals such as HDAC inhibitors but also retained in vivo response to CD40 ligation contributes to improved immune surveillance in these patients which may add to a superior relapse-free survival observed particularly in the presence of other risk factors.

[Expressions of immunogenic molecules in low-dose radiotherapy-treated human renal clear cell carcinoma 786-0 cells].

OBJECTIVE: To explore the effects of low-dose radiation on the expression of immunogenic membrane molecules calreticulin (CRT) and MHC-I/II on the surface of human renal clear cell carcinoma 786-0 cells. METHODS: The inhibitory activity of low-dose radiation on cell line 786-0 was examined by CCK-8 assay. And the post-radiation membrane expressions of CRT, MHC-I and MHC-II were measured by flow cytometry while CRT was visualized by immunofluorescence photography. RESULTS: The inhibition rates on the proliferative capacities of four 786-0 cell lines rose with the incremental radiation doses of 0, 6, 12 and 24 Gy. And the CRT expression levels of each experimental group was significantly higher than that of the control group (P < 0.001). Along with incremental doses of irradiation, the average calreticulin fluorescence intensities increased gradually initially and then there was a downward trend. The membrane expressions of MHC-I and MHC-II of each experimental group was significantly higher than those of the control group (P < 0.05). As the irradiation dose increased, the average MHC-I fluorescence intensities increased gradually in a dose-dependent manner. CONCLUSION: The low-dose radiotherapy may up-regulate CRT and MHC class I/II related with the immunogenicity of tumor cells to induce immune response against tumors.

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

Sequential immunotherapy by vaccination with GM-CSF-expressing glioma cells and CTLA-4 blockade effectively treats established murine intracranial tumors.

Malignant glioma is an incurable disease with a relatively short median survival. Several clinical trials have demonstrated that immunotherapy with vaccination is a safe and possibly effective way of prolonging survival. Antibody-based blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) ligation on T lymphocytes is associated with enhanced antitumor immunity in animal models of cancer and in patients with advanced melanoma. We hypothesized that sequential therapy with granulocyte-macrophage-colony-stimulating factor (GM-CSF)-expressing whole-glioma-cell vaccination and CTLA-4 blockade is an effective strategy for treating established intracranial gliomas. GL261 glioma cells were injected into the right frontal lobes of syngeneic C57/BL6 mice. At days 3, 6, and 9 after tumor implantation, mice were treated with subcutaneous injection of irradiated GM-CSF-expressing GL261 cells. Mice were also treated with intraperitoneal injection of anti-CTLA-4 monoclonal antibodies (mAbs), either at days 3, 6, and 9 or days 12, 15, and 18. Animals were followed for survival. Splenocytes were harvested at day 22 for use in enzyme-linked immunosorbent spot assays. Early treatment of established intracranial gliomas with high-dose CTLA-4 blockade was associated with increased survival in GL261-bearing mice. Later treatment with anti-CTLA-4 monoclonal antibodies did not significantly improve survival compared with control-treated mice. Early vaccination followed by subsequent CTLA-4 blockade was associated with significantly improved survival versus either treatment alone and intensified tumor-specific immunity as measured by interferon-γ enzyme-linked immunosorbent spot assay. Sequential immunotherapy with GM-CSF-expressing irradiated glioma cells and CTLA-4 blockade synergistically prolongs survival in mice bearing established intracranial gliomas.

Suppression of canine myeloid cells by soluble factors from cultured canine tumor cells.

BACKGROUND: Cancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function. METHODS: These studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines. RESULTS: We found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells. CONCLUSION: These results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.

Bacterial immunotherapy of gastrointestinal tumors.

BACKGROUND: Cancer immunotherapy using bacteria dates back over 150 years. The deeper understanding on how the immune system interferes with the tumor microenvironment has led to the re-emergence of bacteria or their related products in immunotherapeutic concepts. In this review, we discuss recent approaches on experimental bacteriolytic therapy, emphasizing the specific interplay between bacteria, immune cells and tumor cells to break the tumor-induced tolerance. RESULTS: Experimental research during the last decades demonstrated beneficial but also adverse influence of bacteria on tumor growth. There is a strong correlation between chronic infections and tumor incidence. However, acute bacterial infections have favourable effects on tumor growth often contributing to complete remission. Tumor regression is usually attributable to both direct tumor cell killing (via apoptosis and/or necrosis, depending on the applied bacteria) and indirect immune stimulation. This includes (I) elimination of immunosuppressive immune cells (i.e. tumor-associated macrophages, myeloid-derived suppressor, and regulatory T cells), (II) suppression of Th2-directed cytokine secretion (TGFα, IL10), (III) providing a pro-inflammatory micro-milieu (tumor infiltrating neutrophils) and (IV) supporting the influx of cytotoxic T cells into tumors. This finally forces the development of an immunological memory and may provide long-term protection against cancer. CONCLUSION: Immunotherapy using bacteria is still a double-edged sword. Experiences from the last years have substantially contributed to when bacteria and defined components thereof might be integrated into immunotherapeutic concepts. Attempts in transferring this approach into the clinics are on their way.

Oxygen is a master regulator of the immunogenicity of primary human glioma cells.

With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (∼20% O2) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (∼5% O2) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O2 more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O2 took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O2 or physiologic 5% O2. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.

Acute lymphoblastic leukemia cells treated with CpG oligodeoxynucleotides, IL-4 and CD40 ligand facilitate enhanced anti-leukemic CTL responses.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the majority of patients initially respond to upfront chemotherapy, relapses with poor prognosis occur in approximately 20% of cases. Thus, novel therapeutic strategies are required to improve long-term survival. B-cell precursor (BCP)-ALL cells express low levels of immunogenic molecules and, therefore, are poorly recognized by the immune system. In the present study, we investigated the effect of various combinations of potent B-cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of primary BCP-ALL cells and a series of BCP-ALL cell lines. The combination of CpG, IL-4 and CD40L was identified as most effective to enhance expression of immunogenic molecules on BCP-ALL cells, resulting in an increased capacity to induce both allogeneic and autologous cytotoxic T lymphocytes (CTL). Importantly, such CTL exhibited significant anti-leukemic cytotoxicity not only towards treated, but also towards untreated BCP-ALL cells. Our results demonstrate that the combination of CpG with other B-cell stimulators is more efficient than CpG alone in generating immunogenic BCP-ALL cells and anti-leukemic CTL. Our results may stimulate the development of novel adoptive T cell transfer approaches for the management of BCP-ALL.

The liposome-incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guéin can directly enhance the susceptibility of cancer cells to lymphokine-activated killer cells through up-regulation of natural-killer group 2, member D ligands.

OBJECTIVE: • To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guéin (BCG)-cell wall skeleton (CWS) treatment. MATERIALS AND METHODS: • The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. • Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. • The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. RESULTS: • Major histocompatibility complex class I-related chain B (MICB) expression was increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. • UL-16-binding protein (ULBP) 1 expression was also increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. • ULBP1 expression was increased ≈2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. • T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an ≈1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an ≈1.4-fold increase at an E : T ratio of 2. CONCLUSIONS: • In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. • T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. • The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.

Test system for trifunctional antibodies in 3D MCTS culture.

The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the trifunctional, bispecific antibody catumaxomab (anti-EpCAM x anti-CD3) or similar therapeutic molecules. The test system is accessible to various analytical methods and thus allows for characterizing multiple parameters, which can help elucidate the mode of action of immunotherapeutic anticancer treatment. For example, the novel approach enables precise, reproducible volume growth analysis of MCTSs under immunotherapeutic treatments. For evaluation of changes within individual spheroids, cryosections can be stained (e.g., for proliferating or apoptotic cells as well as infiltrating PBMCs). Molecular PCR-based assays or flow cytometric analyses allow for discrimination between different cell types, particularly leukocyte subtypes. Furthermore, MCTSs can be disaggregated to form standard monolayers for cell viability or plating efficiency experiments. For these reasons, the MCTS model is a powerful tool to analyze drug efficacy with various endpoints under highly reproducible, standardized conditions.