Assessment of targeted therapy opportunities in sinonasal cancers using patient-derived functional tumor models.

Malignant tumors derived from the epithelium lining the nasal cavity region are termed sinonasal cancers, a highly heterogeneous group of rare tumors accounting for 3 - 5 % of all head and neck cancers. Progress with next-generation molecular profiling has improved our understanding of the complexity of sinonasal cancers and resulted in the identification of an increasing number of distinct tumor entities. Despite these significant developments, the treatment of sinonasal cancers has hardly evolved since the 1980s, and an advanced sinonasal cancer presents a poor prognosis as targeted therapies are usually not available. To gain insights into potential targeted therapeutic opportunities, we performed a multiomics profiling of patient-derived functional tumor models to identify molecular characteristics associated with pharmacological responses in the different subtypes of sinonasal cancer. METHODS: Patient-derived ex vivo tumor models representing four distinct sinonasal cancer subtypes: sinonasal intestinal-type adenocarcinoma, sinonasal neuroendocrine carcinoma, sinonasal undifferentiated carcinoma and SMARCB1 deficient sinonasal carcinoma were included in the analyses. Results of functional drug screens of 160 anti-cancer therapies were integrated with gene panel sequencing and histological analyses of the tumor tissues and the ex vivo cell cultures to establish associations between drug sensitivity and molecular characteristics including driver mutations. RESULTS: The different sinonasal cancer subtypes display considerable differential drug sensitivity. Underlying the drug sensitivity profiles, each subtype was associated with unique molecular features. The therapeutic vulnerabilities correlating with specific genomic background were extended and validated with in silico analyses of cancer cell lines representing different human cancers and with reported case studies of sinonasal cancers treated with targeted therapies. CONCLUSION: The results demonstrate the importance of understanding the differential biology and the molecular features associated with the different subtypes of sinonasal cancers. Patient-derived ex vivo tumor models can be a powerful tool for investigating these rare cancers and prioritizing targeted therapeutic strategies for future clinical development and personalized medicine.

Programmed death-ligand 1 and tumor-infiltrating lymphocytes (TILs) - low TIL density may predict poorer long-term prognosis in T1 laryngeal cancer.

We evaluated the prognostic role of programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in T1 glottic laryngeal squamous cell carcinoma (LSCC). T1 glottic LSCC patients (n = 174) treated at five Finnish university hospitals between 2003 and 2013 were included. Tissue microarray (TMA) blocks were used for PD-L1 immunohistochemistry. TILs were scored from intratumoral and stromal regions in whole tissue sections. Of 174 patients, 92 (53%) had negative, 66 (38%) intermediate, and 16 (9%) high PD-L1 levels. Of 80 patients whose TILs were analyzed, 50 (63%) had low and 30 (38%) high stromal TIL density. Patients with a local recurrence or a new primary tumor of the larynx had lower TIL density than had other patients (p = 0.047). High PD-L1 expression with low stromal TIL density was associated with inferior 5-year disease-specific survival (85% vs. 100%, p = 0.02). In conclusion, in patients treated for T1 glottic LSCC, low stromal TIL density was associated with local recurrences and new primary tumors of the larynx. High PD-L1 expression with low stromal TIL density may be associated with worse survival in T1 glottic LSCC.

The effect of matrices on the gene expression profile of patient-derived head and neck carcinoma cells for in vitro therapy testing.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with a 5-year mortality rate of ~ 50%. New in vitro methods are needed for testing patients' cancer cell response to anti-cancer treatments. We aimed to investigate how the gene expression of fresh carcinoma tissue samples and freshly digested single cancer cells change after short-term cell culturing on plastic, Matrigel or Myogel. Additionally, we studied the effect of these changes on the cancer cells' response to anti-cancer treatments. MATERIALS/METHODS: Fresh tissue samples from HNSCC patients were obtained perioperatively and single cells were enzymatically isolated and cultured on either plastic, Matrigel or Myogel. We treated the cultured cells with cisplatin, cetuximab, and irradiation; and performed cell viability measurement. RNA was isolated from fresh tissue samples, freshly isolated single cells and cultured cells, and RNA sequencing transcriptome profiling and gene set enrichment analysis were performed. RESULTS: Cancer cells obtained from fresh tissue samples changed their gene expression regardless of the culturing conditions, which may be due to the enzymatic digestion of the tissue. Myogel was more effective than Matrigel at supporting the upregulation of pathways related to cancer cell proliferation and invasion. The impacts of anti-cancer treatments varied between culturing conditions. CONCLUSIONS: Our study showed the challenge of in vitro cancer drug testing using enzymatic cell digestion. The upregulation of many targeted pathways in the cultured cells may partially explain the common clinical failure of the targeted cancer drugs that pass the in vitro testing.

Prognostic histological markers in oral tongue squamous cell carcinoma patients treated with (chemo)radiotherapy.

Treatment of oral tongue squamous cell carcinoma (OTSCC) frequently includes surgery with postoperative radiotherapy (RT) or chemoradiotherapy (CRT). Resistance to RT or CRT remains a major clinical challenge and highlights the need to identify predictive markers for it. We included 71 OTSCC patients treated with surgery combined with RT or CRT. We evaluated the association between tumor budding, tumor-stroma ratio (TSR), depth of invasion (DOI), tumor-infiltrating lymphocytes (TILs), hypoxia-inducible factor-1alpha (HIF-1alpha) expression, octamer-binding transcription factor 4 (OCT4) expression, high-endothelial venules (HEVs), and disease-free survival (DFS) using uni- and multivariate analyses. No significant association was observed between the different histological and molecular markers (TSR, DOI, TILs, HEV, HIF-1alph, OCT4) and DFS. However, an associative trend between DOI, budding, and DFS was noted. Further studies with larger cohorts are needed to explore the prognostic value of DOI and budding for OTSCC patients treated with postoperative RT or CRT.

TSPO is a potential independent prognostic factor associated with cellular respiration and p16 in head and neck squamous cell carcinoma.

Background: Treatment resistance and relapse are common problems in head and neck squamous cell carcinoma (HNSCC). Except for p16, no clinically accepted prognostic biomarkers are available for HNSCC. New biomarkers predictive of recurrence and survival are crucial for optimal treatment planning and patient outcome. High translocator protein (TSPO) levels have been associated with poor survival in cancer, but the role of TSPO has not been extensively evaluated in HNSCC. Materials and methods: TSPO expression was determined in a large population-based tissue microarray cohort including 611 patients with HNSCC and evaluated for survival in several clinicopathological subgroups. A TCGA HNSCC cohort was used to further analyze the role of TSPO in HNSCC. Results: TSPO expression was downregulated in more aggressive tumors. Low TSPO expression associated with worse 5-year survival and was an independent prognostic factor for disease-specific survival. Subgroup analyses showed that low TSPO expression associated with worse survival particularly in p16-positive oropharyngeal cancer. In silico analyses supported the prognostic role of TSPO. Cellular respiration had the highest significance in pathway analyses for genes expressed positively with TSPO. Conclusion: Decreased TSPO expression associates with poor prognosis in HNSCC. TSPO is a prognostic biomarker in HNSCC to potentially guide treatment stratification especially in p16-positive oropharyngeal cancer.

Epidemiological Study of p16 Incidence in Head and Neck Squamous Cell Carcinoma 2005-2015 in a Representative Northern European Population.

The incidence of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) has increased globally. Our research goal was to study HNSCC incidence in a representative Northern European population and evaluate the utility of the HPV surrogate marker p16 in clinical decision-making. All new HNSCC patients diagnosed and treated in Southwest Finland from 2005-2015 (n = 1033) were identified and analyzed. During the follow-up period, the incidence of oropharyngeal (OPSCC) and oral cavity squamous cell carcinoma (OSCC) increased, while the incidence of laryngeal squamous cell carcinoma (LSCC) decreased. This clinical cohort was used to generate a population-validated tissue microarray (PV-TMA) archive for p16 analyses. The incidence of p16 positivity in HNSCC and OPSCC increased in southwest Finland between 2005 and 2015. p16 positivity was mainly found in the oropharynx and was a significant factor for improved survival. p16-positive OPSCC patients had a better prognosis, regardless of treatment modality. All HNSCC patients benefited from a combination of chemotherapy and radiotherapy, regardless of p16 expression. Our study reaffirms that p16 expression offers a prognostic biomarker in OPSCC and could potentially be used in cancer treatment stratification. Focusing on p16 testing for only OPSCC might be the most cost-effective approach in clinical practice.

Exposure to alcohol and overall survival in head and neck cancer: A regional cohort study.

BACKGROUND: There is a paucity of knowledge regarding the association of alcohol use with overall survival (OS) of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: All 1033 patients treated for new HNSCC in Southwest Finland regional referral center of Turku University Hospital in 2005-2015. Cox regression analysis was used. Tumor TNM classification, age at baseline and tobacco smoking status were assessed as potential confounders. RESULTS: A history of severe harmful alcohol use with major somatic complications (HR: 1.41; 95%CI: 1.06-1.87; p = 0.017) as well as current use of at least 10 units per week (HR: 1.44, 95%CI: 1.16-1.78; p = 0.001) were associated with OS. CONCLUSIONS: Alcohol consumption of 10-20 units/week, often regarded as moderate use, was found to increase risk of mortality independent of other prognostic variables. Systematic screening of risk level alcohol use and prognostic evaluation of alcohol brief intervention strategies is highly recommended.

Cisplatin overcomes radiotherapy resistance in OCT4-expressing head and neck squamous cell carcinoma.

OBJECTIVES: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patient-selective therapeutic utility. In this study, we examined the role of the stem cell factor OCT4 as a potential biomarker to help clinicians stratify HNSCC patients between radiotherapy and chemoradiotherapy. MATERIALS AND METHODS: OCT4 immunohistochemical staining of a population-validated tissue microarray (PV-TMA) (n = 166) representative of a standard HNSCC patients was carried out, and 5-year survival was analyzed. The results were validated using ex vivo drug sensitivity analysis of HNSCC tumor samples, and further cross-validated in independent oropharyngeal (n = 118), nasopharyngeal (n = 170), and vulvar carcinoma (n = 95) clinical datasets. In vitro, genetically modified, patient-derived HNSCC cells were used. RESULTS: OCT4 expression in HNSCC tumors was associated with radioresistance. However, combination therapy with cisplatin was found to overcome thisradioresistance in OCT4-expressing HNSCC tumors. The results were validated by using several independent patient cohorts. Furthermore, CRISPRa-based OCT4 overexpression in the HNSCC cell line resulted in apoptosis resistance, and cisplatin was found to downregulate OCT4 protein expression in vitro. Ex vivo drug sensitivity analysis of HNSCC tumors confirmed the association between OCT4 expression and cisplatin sensitivity. CONCLUSION: This study introduces OCT4 immunohistochemistry as a simple and cost-effective diagnostic approach for clinical practice to identify HNSCC patients benefitting from radiosensitization by cisplatin using either full or reduced dosing.

T1 glottic laryngeal cancer: the role of routine follow-up visits in detecting local recurrence.

PURPOSE: We assessed the treatment outcome and the benefits of routine follow-up visits in T1 glottic laryngeal squamous cell carcinoma (LSCC). METHODS: Medical records of patients diagnosed with stage T1 glottic LSCC (N = 303) in five Finnish university hospitals between 2003 and 2015 were reviewed. Moreover, data from the Finnish Cancer Registry and the Population Register Center were collected. RESULTS: Of all 38 recurrences, 26 (68%) were detected during a routine follow-up visit, and over half (21 of 38, 55%) presented without new symptoms. Primary treatment method (surgery vs. radiotherapy) was not connected with 5-year disease-specific survival (DSS) or laryngeal preservation rate. CONCLUSION: The majority of recurrences were detected on a routine follow-up visit, and local recurrences often presented without new symptoms. Routine post-treatment follow-up of T1 glottic LSCC seems beneficial. TRIAL REGISTRATION: Trial registration number and date of registration HUS/356/2017 11.12.2017.

Cancer cell line microarray as a novel screening method for identification of radioresistance biomarkers in head and neck squamous cell carcinoma.

BACKGROUND: Currently, no clinically useful biomarkers for radioresistance are available in head and neck squamous cell carcinoma (HNSCC). This study assesses the usefulness of Cell Line Microarray (CMA) method to enhance immunohistochemical screening of potential immunohistochemical biomarkers for radioresistance in HNSCC cell lines. METHODS: Twenty-nine HNSCC cell lines were cultured, cell pellets formalin-fixed, paraffin-embedded, and arrayed. Radioresistance features of the cell lines were combined to immunohistochemical stains for p53, NDFIP1, EGFR, stem cell marker Oct4, and PP2A inhibitor CIP2A. RESULTS: Expression of p53, EGFR or CIP2A did not indicate intrinsic radioresistance in vitro. Stem cell marker Oct4 nuclear positivity and NDFIP1 nuclear positivity was correlated with increased intrinsic radioresistance. CONCLUSION: The usefulness of CMA in analysis of HNSCC cell lines and discovery of biomarkers is demonstrated. CMA is very well adapted to both testing of antibodies in a large panel of cell lines as well as correlating staining results with other cell line characteristics. In addition, CMA-based antibody screening proved an efficient and relatively simple method to identify potential radioresistance biomarkers in HNSCC.

Evaluation of prognostic biomarkers in a population-validated Finnish HNSCC patient cohort.

INTRODUCTION: Prognostic biomarkers and novel therapeutic approaches have been slow to emerge in the treatment of head and neck squamous cell carcinoma (HNSCC). In this study, an HNSCC patient cohort is created and performance of putative prognostic biomarkers investigated in a population-validated setting. The overall goal is to develop a novel way to combine biomarker analyses with population-level clinical data on HNSCC patients and thus to improve the carryover of biomarkers into clinical practice. MATERIALS AND METHODS: To avoid selection biases in retrospective study design, all HNSCC patients were identified and corresponding clinical data were collected from the Southwest Finland geographical area. A particular emphasis was laid on avoiding potential biases in sample selection for immunohistochemical staining analyses. Staining results were evaluated for potential prognostic resolution. RESULTS: After comprehensive evaluation, the patient cohort was found to be representative of the background population in terms of clinical characteristics such as patient age and TNM stage distribution. A negligible drop-out of 1.3% (6/476) was observed during the first follow-up year. By immunohistochemical analysis, the role of previously implicated HNSCC biomarkers (p53, EGFR, p16, CIP2A, Oct4, MET, and NDFIP1) was investigated. DISCUSSION: Our exceptionally representative patient material supports the use of population validation to improve the applicability of results to real-life situations. The failure of the putative prognostic biomarkers emphasizes the need for controlling bias in retrospective studies, especially in the heterogenous tumor environment of HNSCC. The resolution of simple prognostic examination is unlikely to be sufficient to identify biomarkers for clinical practice of HNSCC.

Hepatocyte Growth Factor Receptor overexpression predicts reduced survival but its targeting is not effective in unselected HNSCC patients.

BACKGROUND: MET has emerged as target in head and neck squamous cell carcinoma (HNSCC). However, clinical data on MET inhibition in HNSCC are limited. METHODS: HNSCC biopsies and cell lines were tested for MET activity. The response of cell lines to BAY-853474 was tested in proliferation assays. The prognostic value of MET expression was also analyzed. RESULTS: HNSCC cell lines do not respond to MET inhibition. MET-dependent gastric cancer cell lines have much higher levels of MET expression and phosphorylation than HNSCC cell lines. Clinical samples of HNSCC contain much less MET than responsive models. CONCLUSIONS: No clinical response to MET inhibitors in monotherapy may be expected in unselected cases of HNSCC. Only selected patients with MET amplifications should be treated with MET inhibitors. Patients with increased MET immunoreactivity have shorter overall survival. MET might be useful as marker for the detection of patients with more aggressive types of HNSCC.

PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

Ribosome biogenesis regulates animal growth and is controlled by nutrient-responsive mTOR signaling. How ribosome biogenesis is regulated during the developmental growth of animals and how nutrient-responsive signaling adjusts ribosome biogenesis in this setting have remained insufficiently understood. We uncover PWP1 as a chromatin-associated regulator of developmental growth with a conserved role in RNA polymerase I (Pol I)-mediated rRNA transcription. We further observed that PWP1 epigenetically maintains the rDNA loci in a transcription-competent state. PWP1 responds to nutrition in Drosophila larvae via mTOR signaling through gene expression and phosphorylation, which controls the nucleolar localization of dPWP1. Our data further imply that dPWP1 acts synergistically with mTOR signaling to regulate the nucleolar localization of TFIIH, a known elongation factor of Pol I. Ribosome biogenesis is often deregulated in cancer, and we demonstrate that high PWP1 levels in human head and neck squamous cell carcinoma tumors are associated with poor prognosis.

Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription.

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal-not cancer-stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression.

Potential role for inhibition of protein phosphatase 2A tumor suppressor in salivary gland malignancies.

The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb-Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb-Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome-wide microarray expression analysis, ingenuity pathway analysis, RT-PCR, and immunohistochemistry. Thirty-eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome-wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb-Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb-Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p-S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation.

Copy number increase of oncoprotein CIP2A is associated with poor patient survival in human head and neck squamous cell carcinoma.

BACKGROUND: CIP2A, an inhibitor of PP2A tumour suppressor function, is a widely overexpressed biomarker of aggressive disease and poor therapy response in multiple human cancer types. METHODS: CIP2A and DPPA4 copy number alterations and expression were analysed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in different cell lines and a tissue microarray of 52 HNSCC patients. Results were correlated with patient survival and other clinicopathological data. RESULTS: CIP2A and DPPA4 copy number increase occurred at a relatively high frequency in human HNSCC patient samples. CIP2A but not DPPA4 FISH status was significantly associated with patient survival. CIP2A detection by combining IHC with FISH yielded superior resolution in the prognostication of HNSCC. CONCLUSIONS: CIP2A copy number increase is associated with poor patient survival in human HNSCC. We suggest that the reliability and prognostic value of CIP2A detection can be improved by performing FISH analysis to CIP2A IHC positive tumours.

MYC is not detected in highly proliferating normal spermatogonia but is coupled with CIP2A in testicular cancers.

High MYC expression is linked to proliferative activity in most normal tissues and in cancer. MYC also supports self-renewal and proliferation of many types of tissue progenitor cells. Cancerous inhibitor of PP2A (CIP2A) promotes MYC phosphorylation and activity during intestinal crypt regeneration in vivo and in various cancers. CIP2A also supports male germ cell proliferation in vivo. However, the role of MYC in normal germ cell proliferation and spermatogonial progenitor self-renewal is currently unclear. Here, we demonstrate that male germ cells are CIP2A-positive but lack detectable levels of MYC protein; whereas MYC is highly expressed in Leydig cells and peritubular myoid cells contributing thereby to the testicular stem cell niche. On the other hand, MYC was co-expressed with CIP2A in testicular cancers. These results demonstrate that CIP2A and MYC are spatially uncoupled in the regulation of spermatogenesis, but functional relationship between these two human oncoproteins is established during testicular cancer transformation. We propose that further analysis of mechanisms of MYC silencing in spermatogonial progenitors may reveal novel fundamental information relevant to understanding of MYC expression in cancer.

CIP2A is an Oct4 target gene involved in head and neck squamous cell cancer oncogenicity and radioresistance.

Radiotherapy is a mainstay for treatment of many human cancer types, including head and neck squamous cell carcinoma (HNSCC). Thereby, it is clinically very relevant to understand the mechanisms determining radioresistance. Here, we identify CIP2A as an Oct4 target gene and provide evidence that they co-operate in radioresistance. Oct4 positively regulates CIP2A expression both in testicular cancer cell lines as well as in embryonic stem cells. To expand the relevance of these findings we show that Oct4 and CIP2A are co-expressed in CD24 positive side-population of patient-derived HNSCC cell lines. Most importantly, all Oct4 positive HNSCC patient samples were CIP2A positive and this double positivity was linked to poor differentiation level, and predicted for decreased patient survival among radiotherapy treated HNSCC patients. Oct4 and CIP2A expression was also linked with increased aggressiveness and radioresistancy in HNSCC cell lines. Together we demonstrate that CIP2A is a novel Oct4 target gene in stem cells and in human cancer cell lines. Clinically these results suggest that diagnostic evaluation of HNSCC tumors for Oct4 or Oct4/CIP2A positivity might help to predict HNSCC tumor radioresistancy. These results also identify both Oct4 and CIP2A as potential targets for radiosensitation.

Reconstruction of mouse testicular cellular microenvironments in long-term seminiferous tubule culture.

Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1) and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA) and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger), LIN28, Gpr125 (G protein-coupled receptor 125), CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs) that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH) which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2-4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to establish in vitro conditions that allow spontaneous reconstruction of testicular cellular microenvironments.