Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment.

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Reduction of daily-use parabens and phthalates reverses accumulation of cancer-associated phenotypes within disease-free breast tissue of study subjects.

Estrogenic overstimulation is carcinogenic to the human breast. Personal care products (PCPs) commonly contain xenoestrogens (XE), such as parabens and phthalates. Here, we identified the adverse effects of persistent exposure to such PCPs directly within human estrogen responsive breast tissue of subjects enrolled in a regimen of reduced XE use (REDUXE). Pre- and post-intervention fine needle aspirates (FNAs) of the breast were collected from healthy volunteers who discontinued the use of paraben and phthalate containing PCPs over a 28 d period. Based on high-dimensional gene expression data of matched FNA pairs of study subjects, we demonstrate a striking reversal of cancer-associated phenotypes, including the PI3K-AKT/mTOR pathway, autophagy, and apoptotic signaling networks within breast cells of REDUXE compliant subjects. These, and other altered phenotypes were detected together with a significant reduction in urinary parabens and phthalate metabolites. Moreover, in vitro treatment of paired FNAs with 17ß-estradiol (E2), displayed a 'normalizing' impact of REDUXE on gene expression within known E2-modulated pathways, and on functional endpoints, including estrogen receptor alpha: beta ratio, and S-phase fraction of the cell cycle. In a paradigm shifting approach facilitated by community-based participatory research, REDUXE reveals unfavorable consequences from exposure to XEs from daily-use PCPs. Our findings illustrate the potential for REDUXE to suppress pro-carcinogenic phenotypes at the cellular level towards the goal of breast cancer prevention.

Protein Profiling of Breast Carcinomas Reveals Expression of Immune-Suppressive Factors and Signatures Relevant for Patient Outcome.

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification.

[Patient-derived microtumors : Potential for therapeutic response prediction-a case study]. / Patientenabgeleitete Mikrotumoren : Eine Möglichkeit der Vorhersage des medikamentösen Therapieansprechens ­ ein Fallbericht.

BACKGROUND: In view of continued development of new oncological approaches, there is a high demand for personalized tumor therapy. However, fast and effective functional platforms for the prediction of individual patient response to drug therapy are largely unavailable. Various promising approaches have already been described for three-dimensional cell culture models, which represent cellular complexity and almost identical structures of the original tumor tissue. OBJECTIVES: Based on a case report, we show the capability and results of a novel test system using patient-derived microtumors (PDMs) and autologous tumor-infiltrating lymphocytes (TILs) for the prediction of response to cancer therapy. METHODS: We established PDMs and TILs from primary tumor tissue of a renal cell carcinoma metastasis. Using immunohistochemistry and multiplex florescence-activated cell sorting (FACS ) analyses, the PDMs and TILs were characterized regarding to histology and immunophenotype. Tumor-specific cytotoxicity of standard of care and investigational compounds were assessed. The results were compared to the patient's individual in vivo response to therapy. CONCLUSION: The cytotoxicity assay of PDMs and TILs showed a significant therapeutic response (p = 0.0004) to therapy with a programmed cell death protein 1 (PD-1) inhibitor and lenvatinib compared to the control. The in vitro results correlated positively with the in vivo data. In the future, patient-derived models could predict response to cancer therapy and may help to optimize treatment decision-making.

A Platform of Patient-Derived Microtumors Identifies Individual Treatment Responses and Therapeutic Vulnerabilities in Ovarian Cancer.

In light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease, we developed a 3D preclinical tumor model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) to identify individual treatment vulnerabilities and validate chemo-, immuno- and targeted therapy efficacies. Enzymatic digestion of primary ovarian cancer tissue and cultivation in defined serum-free media allowed rapid and efficient recovery of PDM, while preserving histopathological features of corresponding patient tumor tissue. Reverse-phase protein array (RPPA)-analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Co-cultures of PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Combining protein pathway analysis and drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision making.

Generation and characterization of the human induced pluripotent stem cell line NMIi010-A from peripheral blood mononuclear cells of a healthy 49-year old male individual.

Peripheral-blood derived CD34+ hematopoietic stem and progenitor cells were isolated from a 49-year old male donor and were successfully reprogrammed into human induced pluripotent stem cells (hiPSCs) using integration-free episomal vectors. The hiPSC line exhibited a typical stem cell-like morphology and endogenously expressed several pluripotency markers by concomitant loss of exogenous reprogramming vectors. Genomic integrity was confirmed by microarray-based comparative genomic hybridization (array CGH). Further analysis affirmed the ability of this hiPSC line to differentiate into all three germ layers. Thus, the reported cell line may serve as a healthy control for disease modeling.

Targeting CSF1R Alone or in Combination with PD1 in Experimental Glioma.

Glioblastoma is an aggressive primary tumor of the central nervous system. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting therapeutic approach. Tumor-associated macrophages represent an abundant population of tumor-infiltrating host cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We investigated combination treatments of anti-CSF1R alone or in combination with anti-PD1 antibodies in an orthotopic syngeneic glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies led to longterm survivors in vivo. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Our results identify CSF1R as a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.