OrganoIDNet: a deep learning tool for identification of therapeutic effects in PDAC organoid-PBMC co-cultures from time-resolved imaging data.

PURPOSE: Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required. METHODS: We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo. RESULTS: Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added. CONCLUSION: Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.

Diagnostic leukapheresis reveals distinct phenotypes of NSCLC circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs. METHODS: DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations. RESULTS: Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes. CONCLUSIONS: In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.

Colorectal cancer-associated fibroblasts inhibit effector T cells via NECTIN2 signaling.

Cancer-associated fibroblasts play a crucial role within the tumor microenvironment. However, a comprehensive characterization of CAF in colorectal cancer (CRC) is still missing. We combined scRNA-seq and spatial proteomics to decipher fibroblast heterogeneity in healthy human colon and CRC at high resolution. Analyzing nearly 23,000 fibroblasts, we identified 11 distinct clusters and verified them by spatial proteomics. Four clusters, consisting of myofibroblastic CAF (myCAF)-like, inflammatory CAF (iCAF)-like and proliferating fibroblasts as well as a novel cluster, which we named "T cell-inhibiting CAF" (TinCAF), were primarily found in CRC. This new cluster was characterized by the expression of immune-interacting receptors and ligands, including CD40 and NECTIN2. Co-culture of CAF and T cells resulted in a reduction of the effector T cell compartment, impaired proliferation, and increased exhaustion. By blocking its receptor interaction, we demonstrated that NECTIN2 was the key driver of T cell inhibition. Analysis of clinical datasets showed that NECTIN2 expression is a poor prognostic factor in CRC and other tumors. In conclusion, we identified a new class of immuno-suppressive CAF with features rendering them a potential target for future immunotherapies.

Endogenous Signaling Molecule Activating (ESMA) CARs: A Novel CAR Design Showing a Favorable Risk to Potency Ratio for the Treatment of Triple Negative Breast Cancer.

As chimeric antigen receptor (CAR) T cell therapy continues to gain attention as a valuable treatment option against different cancers, strategies to improve its potency and decrease the side effects associated with this therapy have become increasingly relevant. Herein, we report an alternative CAR design that incorporates transmembrane domains with the ability to recruit endogenous signaling molecules, eliminating the need for stimulatory signals within the CAR structure. These endogenous signaling molecule activating (ESMA) CARs triggered robust cytotoxic activity and proliferation of the T cells when directed against the triple-negative breast cancer (TNBC) cell line MDA-MB-231 while exhibiting reduced cytokine secretion and exhaustion marker expression compared to their cognate standard second generation CARs. In a NOD SCID Gamma (NSG) MDA-MB-231 xenograft mouse model, the lead candidate maintained longitudinal therapeutic efficacy and an enhanced T cell memory phenotype. Profound tumor infiltration by activated T cells repressed tumor growth, further manifesting the proliferative capacity of the ESMA CAR T cell therapy. Consequently, ESMA CAR T cells entail promising features for improved clinical outcome as a solid tumor treatment option.

Culture expansion of CAR T cells results in aberrant DNA methylation that is associated with adverse clinical outcome.

Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.

Targeting Stage-Specific Embryonic Antigen 4 (SSEA-4) in Triple Negative Breast Cancer by CAR T Cells Results in Unexpected on Target/off Tumor Toxicities in Mice.

Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

Basal-like mammary carcinomas stimulate cancer stem cell properties through AXL-signaling to induce chemotherapy resistance.

Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.

A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level.

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (± IL-2) or control BDCA-2 CAR T cell (± IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D µCT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D µCT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

Enhancing the Efficacy of CAR T Cells in the Tumor Microenvironment of Pancreatic Cancer.

Pancreatic cancer has the worst prognosis and lowest survival rate among all types of cancers and thus, there exists a strong need for novel therapeutic strategies. Chimeric antigen receptor (CAR)-modified T cells present a new potential option after successful FDA-approval in hematologic malignancies, however, current CAR T cell clinical trials in pancreatic cancer failed to improve survival and were unable to demonstrate any significant response. The physical and environmental barriers created by the distinct tumor microenvironment (TME) as a result of the desmoplastic reaction in pancreatic cancer present major hurdles for CAR T cells as a viable therapeutic option in this tumor entity. Cancer cells and cancer-associated fibroblasts express extracellular matrix molecules, enzymes, and growth factors, which can attenuate CAR T cell infiltration and efficacy. Recent efforts demonstrate a niche shift where targeting the TME along CAR T cell therapy is believed or hoped to provide a substantial clinical added value to improve overall survival. This review summarizes therapeutic approaches targeting the TME and their effect on CAR T cells as well as their outcome in preclinical and clinical trials in pancreatic cancer.

CD49b, CD87, and CD95 Are Markers for Activated Cancer-Associated Fibroblasts Whereas CD39 Marks Quiescent Normal Fibroblasts in Murine Tumor Models.

Fibroblasts are thought to be key players in the tumor microenvironment. Means to identify and isolate fibroblasts as well as an understanding of their cancer-specific features are essential to dissect their role in tumor biology. To date, the identification of cancer-associated fibroblasts is widely based on generic markers for activated fibroblasts in combination with their origin in tumor tissue. This study was focused on a deep characterization of the cell surface marker profile of cancer-associated fibroblasts in widely used mouse tumor models and defining aberrant expression profiles by comparing them to their healthy counterparts. We established a generic workflow to isolate healthy and cancer-associated fibroblasts from solid tissues, thereby reducing bias, and background noise introduced by non-target cells. We identified CD87, CD44, CD49b, CD95, and Ly-6C as cancer-associated fibroblast cell surface markers, while CD39 was identified to mark normal fibroblasts from healthy tissues. In addition, we found a functional association of most cancer-related fibroblast markers to proliferation and a systemic upregulation of CD87, and CD49b in tumor-bearing mice, even in non-affected tissues. These novel markers will facilitate the characterization of fibroblasts and shed further light in their functions and implication in cancer progression.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Cellular Prion Protein PrPC and Ecto-5'-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays(1). Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research (2-4). Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors(4) or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable. Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis (5). To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Comparison between cold plasma, electrochemotherapy and combined therapy in a melanoma mouse model.

The study was undertaken to compare antitumor efficacy of electrochemotherapy (ECT) with cold plasma therapy (CP) in a melanoma mouse model. After melanoma implantation into the flank of C57BL/6N mice, CP by two different plasma sources (APPJ and DBD) was applied directly to the tumor surface. ECT was performed with bleomycin intravenously at a field strength of 1000 V/cm without or combined with CP. Primary endpoints were tumor growth acceleration (TGA), daily volume progression (DVP) and survival after treatment. Both plasma sources as single treatment showed a significant TGA delay, which proved less effective than ECT. CP (APPJ) combined with ECT (ECJ) significantly improved per cent mouse survival, with significant superiority compared with ECT. Plasma therapy alone albeit less effective seems a potential alternative to ECT in patients with melanoma and can be applied manifold in a session without general anaesthesia. Accordingly, CP alone and combined with ECT may serve as new option in palliative skin melanoma therapy.

Highly sensitive profiling of CD44+/CD24- breast cancer stem cells by combining global mRNA amplification and next generation sequencing: evidence for a hyperactive PI3K pathway.

We performed next generation sequencing- and microarray-based gene expression profiling of CD44(+)/CD24(-)/CD45(-) breast CSCs (cancer stem cells) isolated from primary ERα-positive breast cancer. By combining semi-automated dissociation of human tumor tissue, magnetic cell sorting and cDNA amplification less than 500 CSCs were required for transcriptome analyses. Besides overexpressing genes involved in maintenance of stemness, the CSCs showed higher levels of genes that drive the PI3K pathway, including EGFR, HB-EGF, PDGFRA/B, PDGF, MET, PIK3CA, PIK3R1 and PIK3R2. This suggests that, in CSCs of ERα-positive breast cancer, the PI3K pathway which is involved in endocrine resistance is hyperactive.