Stromal Composition Research Articles

Distinguishing Tumor and Stromal Sources of MicroRNAs Linked to Metastasis in Cutaneous Melanoma

MicroRNA (miRNA) dysregulation in cancer causes changes in gene expression programs regulating tumor progression and metastasis. Candidate metastasis suppressor miRNA are often identified by differential expression in primary tumors compared to metastases. Here, we performed comprehensive analysis of miRNA expression in The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) tumors (97 primary, 350 metastatic), and identified candidate metastasis-suppressor miRNAs. Differential expression analysis revealed miRNA significantly downregulated in metastatic tumors, including miR-205, miR-203, miR-200a-c, and miR-141. Furthermore, sequential feature selection and classification analysis identified miR-205 and miR-203 as the miRNA best able to discriminate between primary and metastatic tumors. However, cell-type enrichment analysis revealed that gene expression signatures for epithelial cells, including keratinocytes and sebocytes, were present in primary tumors and significantly correlated with expression of the candidate metastasis-suppressor miRNA. Examination of miRNA expression in cell lines revealed that candidate metastasis-suppressor miRNA identified in the SKCM tumors, were largely absent in melanoma cells or melanocytes, and highly restricted to keratinocytes and other epithelial cell types. Indeed, the differences in stromal cell composition between primary and metastatic tumor tissues is the main basis for identification of differential miRNA that were previously classified as metastasis-suppressor miRNAs. We conclude that future studies must consider tumor-intrinsic and stromal sources of miRNA in their workflow to identify bone fide metastasis-suppressor miRNA in cutaneous melanoma and other cancers.

Histopathological prognostic markers in metastatic lung adenocarcinoma, validation of stromal fibrosis and immunoscore as easy to interpret findings and their correlation with survival.

e21566 Background: Several histopathological markers have been associated with immune system activity and its relationship with clinical outcomes in lung cancer. Determination of tumor infiltrating lymphocytes as well as specific subpopulations could reveal cytotoxic potential. Additionally, changes in stromal composition and fibrosis could also reflect potential cellular interactions with the tumor lesions. In this study we validate these two findings as potential prognostic factors. Methods: A retrospective cohort analysis of patients with advanced lung adenocarcinoma treated in a reference center in Bogota, Colombia was conducted. Demographic as well as treatment variables were collected between January 2018 and December 2019. In addition to clinical outcomes, the percentage of stromal fibrosis and immunoscore were estimated for all biopsy samples by a trained oncological pathologist. Results: A total of 133 patients were included. Male predominance (51.1%) with Karnofsky performance scores (KPS) greater than 80 and 19% of prior cigarette exposure (26 patients) was identified in the cohort. Median overall survival of the cohort was 27.1 months (95%CI 22.1-NA) and progression free survival of 19.4 months (95%CI 18-24.5 months). Percentage of fibrous stroma and immunoscore were associated with a benefit in both OS and PFS. For patients who presented with a low stromal fibrosis, defined as harboring less than 10%, OS reached a median of 25.1 months (95%CI 21.5- 40.5 months) compared to 38.7 months for patients with high fibrosis (95%CI 27.7 months – NA), (p = 0.02). In terms of immunoscore, values under 40% were associated with a longer median OS (38.7 months, [95%CI 31.3- 58.6 months] vs 22.1 months [95%CI19.8- 42.3 months], p < 0.001). Immunoscore and stromal fibrosis were not codependent variables (p < 0.001), suggesting that these markers offer independent prognostic value with regards to OS. Conclusions: Stromal fibrosis and immunoscore serve as prognostic factors that could be easily interpreted and evaluated in order to offer prognostic classification of patients with metastatic adenocarcinoma of the lung.

Effect of general anesthetics on the tumor micro-environment in mouse models of breast cancers of spontaneous metastasis.

e15503 Background: Metastatic breast cancer is a pressing health concern worldwide. Various treatments have been developed but no significant long-term changes in overall survival are observed. Therefore, there is a demand to improve current therapies to treat this disease. Surgical resection of the primary tumors is essential in the treatment. However, accumulating evidence alludes to a role for volatile anesthetics which are used during the surgery in metastatic tumor development, but the mechanism remains largely unknown. We have shown anesthetics exert different effects on lung metastasis in mouse models of breast cancers. This study analyses the effect of general anesthetics in lung microenvironment associated with the increased metastases. Methods: Balb/c mice and NOD-SCID mice were orthotopically implanted with 4T1 cells and MDA-MB-231 cells respectively, in the mammary fat pad to generate primary tumors. Mice were subjected to the tested anesthetic during implantation and/or before and after surgery. Surgical dissection of primary tumor was performed under anesthesia with sevoflurane or an intravenous anesthetic propofol. Survival curve was constructed and analysed. Mice were euthanized to harvest tissues for histology and cell analysis. Results: As we previously reported, surgical dissection of primary tumor in mice under anesthesia with sevoflurane led to significantly more lung metastasis than with propofol in both syngeneic murine 4T1 and xenograft human MDA-MB-231 breast cancer models. Sevoflurane was associated with increased IL6(Li, Huang, & Lin, 2020). Here we show that anesthesia with sevoflurane resulted in changes of stroma composition in the lung, which was reversed by IL6 pathway interruption. Conclusions: Those results contribute to our understanding of effects of sevoflurane on cancer metastasis and suggest a potential therapeutic approach to overcome the risk of general anesthesia. Li, R., Huang, Y., & Lin, J. (2020). Distinct effects of general anesthetics on lung metastasis mediated by IL-6/JAK/STAT3 pathway in mouse models. Nat Commun, 11, 642.

The prognostic significance of immune microenvironment in breast ductal carcinoma in situ

BackgroundThe role of different subtypes of tumour infiltrating lymphocytes (TILs) in breast ductal carcinoma in situ (DCIS) is still poorly defined. This study aimed to assess the prognostic significance of B and T lymphocytes and immune checkpoint proteins expression in DCIS.MethodsA well characterised DCIS cohort (n = 700) with long-term follow-up comprising pure DCIS (n = 508) and DCIS mixed with invasive carcinoma (IBC; n = 192) were stained immunohistochemically for CD20, CD3, CD4, CD8, FOXP3, PD1 and PDL1. Copy number variation and TP53 mutation status were assessed in a subset of cases (n = 58).ResultsCD3+ lymphocytes were the predominant cell subtype in the pure DCIS cohort, while FOXP3 showed the lowest levels. PDL1 expression was mainly seen in the stromal TILs. Higher abundance of TILs subtypes was associated with higher tumour grade, hormone receptor negativity and HER2 positivity. Mutant TP53 variants were associated with higher levels of stromal CD3+, CD4+ and FOXP3+ cells. DCIS coexisting with invasive carcinoma harboured denser stromal infiltrates of all immune cells and checkpoint proteins apart from CD4+ cells. Stromal PD1 was the most differentially expressed protein between DCIS and invasive carcinoma (Z = 5.8, p < 0.0001). Dense TILs, stromal FOXP3 and PDL1 were poor prognostic factors for DCIS recurrence, while dense TILs were independently associated with poor outcome for all recurrences (HR = 7.0; p = 0.024), and invasive recurrence (HR = 2.1; p = 0.029).ConclusionsImmunosuppressive proteins are potential markers for high risk DCIS and disease progression. Different stromal and intratumoural lymphocyte composition between pure DCIS, DCIS associated with IBC and invasive carcinoma play a potential role in their prognostic significance and related to the underlying genomic instability. Assessment of overall TILs provides a promising tool for evaluation of the DCIS immune microenvironment.

Breast Mammographic Density: Stromal Implications on Breast Cancer Detection and Therapy

Current evidences state clear that both normal development of breast tissue as well as its malignant progression need many-sided local and systemic communications between epithelial cells and stromal components. During development, the stroma, through remarkably regulated contextual signals, affects the fate of the different mammary cells regarding their specification and differentiation. Likewise, the stroma can generate tumour environments that facilitate the neoplastic growth of the breast carcinoma. Mammographic density has been described as a risk factor in the development of breast cancer and is ascribed to modifications in the composition of breast tissue, including both stromal and glandular compartments. Thus, stroma composition can dramatically affect the progression of breast cancer but also its early detection since it is mainly responsible for the differences in mammographic density among individuals. This review highlights both the pathological and biological evidences for a pivotal role of the breast stroma in mammographic density, with particular emphasis on dense and malignant stromas, their clinical meaning and potential therapeutic implications for breast cancer patients.

Abstract P1-03-02: Histoepigenetic characterization of breast cancer patient derived xenografts (PDX) implicates epithelial-stromal interactions in differential chemotherapy resistance and PDX engraftment

Abstract Background: Epithelial-stromal interactions play a critical role in treatment resistance, and the state of the stroma can be an indicator for a tumor’s resistance profile. We have developed and characterized a large group of breast cancer PDX models (n=69) (Zhang et al.,2013) and have imported PDX from other sources for inclusion into preclinical drug testing trials. These PDX represent the three main subtypes defined clinically by IHC/FISH (i.e. Estrogen Receptor positive, HER2 positive, and “Triple-negative”). Because the epithelium and stroma in PDX are from two different species (human and mouse, respectively) mRNA (and protein) expression levels can be assigned in a species-specific manner and interrogated computationally to divine interactions that may be important for PDX behaviors including treatment response or engraftment behavior. Methods: Fifty “triple negative” PDX were treated with four weekly cycles of either docetaxel (20mg/kg, IP) or carboplatin (50mg/kg, IP) vs. control, and evaluated quantitatively for the change in tumor volume from baseline (~200mm3) after four weeks. Using deep RNA-seq data (~200M reads/sample), we identified interactions between the epithelial and stromal in the PDX models using CASTIN (Komura et al.,2016) and variations in cancer specific pathways. We then employed a machine learning approach to identify ligand-receptor interactions whose differential expression correlated with treatment response to each agent. We also used epigenomic deconvolution (Onuchic et al., 2016) to devise a novel classification method based on the cancer-cell specific epigenomic profiles of TCGA samples, and then used it to classify the PDX with the intent to correlate with treatment response. Results: PDX showed a full range of responses to each agent, from total resistance to complete response. However, several PDX showed differential responses to either docetaxel or carboplatin. We identified variation in the expression patterns of cancer-associated pathways in the epithelial cell fraction of the tumors, including Wnt signaling, as well as pathways involved in hepatic fibrosis. Informative interactions included bidirectional Eph receptor-ephrin signaling which has previously been found to be overexpressed in breast cancer (Vaught et al.,2008). In the epigenomic deconvolution analysis, our classification scheme divided the basal-like samples in TCGA into two groups: a “hot” immune profile-enriched group, and a “cold” or immune profile-deficient group. By applying this classification to the PDX models, unexpectedly, an overwhelming majority of the triple-negative PDX fell into the “cold” basal group. Thus, classification in this manner showed no association with treatment response in the PDX. Conclusions: These results indicate that tumors may respond uniquely to a given chemotherapeutic, and suggest that differential expression of signaling pathways as well as specific ligand-receptor pairs may prove predictive of resistance and could allow for the development of novel therapies targeting these tissue interactions. While our epigenomic deconvolution results showed no association with treatment response, they unexpectedly suggested that success of PDX engraftment may depend on the stromal immune system composition of the primary tumor such that “hot” primary tumors have lower engraftment efficiency in the “cold” stromal environment of immunocompromised mice. Citation Format: Varduhi Petrosyan, Chen Huang, Ramakrishnan R Srinivasan, Lacey E Dobrolecki, Christina Sallas, Alaina N Lewis, Tao Wang, Bing Zhang, Aleksandar Milosavljevic, Michael T Lewis. Histoepigenetic characterization of breast cancer patient derived xenografts (PDX) implicates epithelial-stromal interactions in differential chemotherapy resistance and PDX engraftment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-02.

A 3-step Mechanical Digestion Method to Harvest Adipose-derived Stromal Vascular Fraction.

Adipose stromal vascular fraction (SVF) isolation with enzymatic digestion is the gold standard, but is expensive, having practical and legal concerns. The alternative mechanical SVF isolation methods provide lower cell yields as they employ either centrifugation, emulsification, or digestion steps alone. We combined mechanical processing with buffer incubation and centrifugation steps into an isolation method called "mechanical digestion" and compared the cell yields with that of enzymatic digestion. A total of 40-mL lipoaspirate was harvested from 35 women undergoing liposuction and was submitted to conventional enzymatic digestion for SVF isolation or mechanical digestion using a closed unit harnessing 3 ports with blades, followed by buffer incubation and centrifugation. Culture of the SVFs and flow cytometry were performed. The SVF cell yield obtained by enzymatic digestion was significantly higher 3.38 × 106/mL (±3.63; n = 35) than that obtained by mechanical digestion 1.34 × 106/mL (±1.69; n = 35), P = 0.015. The average cell viability was 82.86% ± 10.68 after enzymatic digestion versus 85.86% ± 5.74 after mechanical digestion, which was not significant. Mechanical digested SVF expressed 2-fold higher stem cell surface markers compared with enzymatically digested SVF. Mechanical digestion was less time consuming, cost effective, and did not require a specific laboratory environment. Mechanically digested SVF was comparable to enzymatically digested SVF in terms of stromal cell composition and viability. With mechanical digestion, we can isolate 30%-50% SVF cells of that isolated with enzymatic digestion. Further studies are warranted to determine the clinical outcomes.

B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

TMOD-36. DEVELOPMENT OF A PATIENT-DERIVED ORTHOTOPIC XENOGRAFT GLIOBLASTOMA MODEL PHENOTYPICALLY REPRESENTATIVE OF THE CLINICAL FEATURES

Abstract In murine models, patient derived orthotopic xenografts (PDOXs) generated from neurosphere cultures of human glioblastomas (GBMs) are unmatched in representing the molecular heterogeneity of the disease. However, intra-axial tumors with blood-brain barrier (BBB) breakdown visible on magnetic resonance imaging (MRI) are rare in these models and, thus, phenotypic recapitulation of human tumors is typically absent. We hypothesized that this absence lay in the interaction between tumor and stroma and that manipulation of the tumor stromal composition might produce a consistent BBB breakdown. Thirty-two athymic nude rats were implanted intracerebrally with one of two neurosphere cell lines (HF2303 or HF3016) derived from the tumors of two GBM patients demonstrating post-gadolinium enhancement on MRI. Neurospheres (3x105 cells in 10μl) were co-injected with a preparation containing either 5μl Matrigel with high (M+) or low (M-) growth factors, or PBS (M0). When the lesion reached 4 to 5 mm in diameter on imaging, MRI with contrast was used to study BBB breakdown. Two animals with only extra-axial tumors were excluded from analysis, and the intra-axial tumors were graded for radiological evidence of BBB breakdown on a scale of 0 to 4, with 0 being no evidence, and 4 being florid enhancement. A two-way ordinal logistic regression model was fit with treatment factors (three levels: M0, M+ and M-) and cell line (two levels: HF2303 and HF3016). A Wilcoxon Rank Sum test analysis within each cell line demonstrated a significant BBB breakdown effect with Matrigel in HF3016 (N=16, p=0.004), but not for HF2303 (N=16, p=0.328). By producing phenotypic conditions closer to the human glioma pathology, a reliable BBB breakdown in PDOX models represents a significant advance in assessing experimental therapies. However, as demonstrated herein, the effect of stromal enhancement is cell-line dependent, and the biological correlates that accompany BBB breakdown still need to be described.

Development of a predictive model for stromal content in prostate cancer samples to improve signature performance.

Prostate cancer is heterogeneous in both cellular composition and patient outcome, and development of biomarker signatures to distinguish indolent from aggressive tumours is a high priority. Stroma plays an important role during prostate cancer progression and undergoes histological and transcriptional changes associated with disease. However, identification and validation of stromal markers is limited by a lack of datasets with defined stromal/tumour ratio. We have developed a prostate‐selective signature to estimate the stromal content in cancer samples of mixed cellular composition. We identified stromal‐specific markers from transcriptomic datasets of developmental prostate mesenchyme and prostate cancer stroma. These were experimentally validated in cell lines, datasets of known stromal content, and by immunohistochemistry in tissue samples to verify stromal‐specific expression. Linear models based upon six transcripts were able to infer the stromal content and estimate stromal composition in mixed tissues. The best model had a coefficient of determination R 2 of 0.67. Application of our stromal content estimation model in various prostate cancer datasets led to improved performance of stromal predictive signatures for disease progression and metastasis. The stromal content of prostate tumours varies considerably; consequently, deconvolution of stromal proportion may yield better results than tumour cell deconvolution. We suggest that adjusting expression data for cell composition will improve stromal signature performance and lead to better prognosis and stratification of men with prostate cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

POU1F1 transcription factor promotes breast cancer metastasis via recruitment and polarization of macrophages.

Cancer progression requires cells surrounding tumors be reeducated and activated to support tumor growth. Oncogenic signals from malignant cells directly influence stromal composition and activation, but the factors mediating this communication are still not well understood. We have previously shown that the transcription factor POU class 1 homeobox 1 (POU1F1), also known as Pit-1, induces profound changes on neoplastic cell-autonomous processes favoring metastasis in human breast cancer. Here we describe for the first time Pit-1-mediated paracrine actions on macrophages in the tumor microenvironment by using cell lines in vitro, zebrafish and mouse models in vivo, and samples from human breast cancer patients. Through the release of CXCL12, Pit-1 in tumor cells was found to mediate the recruitment and polarization of macrophages into tumor-associated macrophages (TAMs). In turn, TAMs collaborated with tumor cells to increase tumor growth, angiogenesis, extravasation and metastasis to lung. Our data reveal a new mechanism of cooperation between tumor cells and macrophages favoring metastasis and poor clinical outcome in human breast cancer, which suggests that Pit-1 and CXCL12 should be further studied as potential prognostic and therapeutic indicators. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Global Transcriptomic Profiling of the Bone Marrow Stromal Microenvironment During Postnatal Development, Aging and Inflammation

Bone marrow (BM) stromal cells provide the structural and regulatory framework for hematopoiesis and contribute to developmental-stage specific niches, such as those preserving hematopoietic stem cell (HSCs). Despite recent advances in our understanding of stromal composition and function, little is known on the dynamic transcriptional remodeling that this compartment undergoes over time and during adaptation to stress. Similarly, how molecular changes in stroma are linked to age-related modulation of hematopoiesis is poorly understood. Using RNA-sequencing, we performed a longitudinal comparison of the transcriptional profile of four principal mesenchymal and endothelial stromal subsets, namely CXCL12-abundant reticular (CARc), PDGFR-α+Sca-1+, sinusoidal (SECs) and arterial endothelial cells (AECs), isolated from early postnatal, adult and aged mice. Our data i) provide molecular fingerprints defining novel, cell-specific anatomical and functional features ii) reveal radical reprogramming of pro-hematopoietic, immune and matrisomic transcriptional programs during the narrow temporal transition from juvenile to adult stages iii) demonstrate that homeostatic aging is characterized by a progressive and pronounced upregulation of pro-inflammatory gene-expression and loss of stromal cell fitness. By profiling in vivo responses of stromal cells to infection-mimicking agents, we finally demonstrate that transcriptomic pathways elicited by sterile inflammation are largely recapitulated during aging, thereby supporting the inflammatory basis of aging-related adaptations of BM hematopoietic function.

A Clinically Applicable Gene-Expression Classifier Reveals Intrinsic and Extrinsic Contributions to Consensus Molecular Subtypes in Primary and Metastatic Colon Cancer.

Four consensus molecular subtypes (CMS1-4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer-related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). We developed a colorectal cancer-focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.

Fibroblast as a critical stromal cell type determining prognosis in prostate cancer.

BackgroundTumor stroma associates with prostate cancer (PCa) progression, but its specific cellular composition and association to patient survival outcome have not been characterized.MethodsWe analyzed stromal composition in human PCa using multiplex immunohistochemistry and quantitative, high‐resolution image analysis in two retrospective, formalin‐fixed paraffin embedded observational clinical cohorts (Cohort I, n = 117; Cohort II, n = 340) using PCa‐specific mortality as outcome measurement.ResultsA high proportion of fibroblasts associated with aggressive disease and castration‐resistant prostate cancer (CRPC). In a multivariate analysis, increase in fibroblast proportion predicted poor cancer‐specific outcome independently in the two clinical cohorts studied.ConclusionsFibroblasts were the most important cell type in determining prognosis in PCa and associated with CRPC. Thus, the stromal composition could be critically important in developing diagnostic and therapeutic approaches to aggressive prostate cancer.

Abstract 2887: Patient-derived 3-dimensional models of pediatric tumors for in situ study of cancer biology, immunotherapy, and drug treatment

Abstract The 2D culture systems are currently the mainstay of pre-clinical testing of potential therapeutics, however, these models fail to recapitulate the dynamic and heterogenous conditions found within the in vivo tumor microenvironment and are poor predictors of clinical activity. Patient derived xenograft models of cancer excel recapitulating these complexities but are not the most tractable approaches for high throughput efficacy testing, particularly of immuno-oncologic therapies. In addition, small animal imaging techniques to noninvasively and longitudinally monitor PDX tumor growth require costly equipment and a high level of technical expertise. Thus, there is a tremendous need for in vitro model systems with improved diagnostic accuracy to better predict therapeutic responses earlier in drug development. This need is even more acute for pediatric cancers such as osteosarcoma (OS) and glioblastoma (GB), for which there are few or no viable non-clinical models to investigate basic science or for pre-clinical drug efficacy studies.We have developed a 3D culture system integrating 3D printing in Liquid Like Solids (LLS) materials to enable the precise arrangement of delicate and highly detailed assemblies of cells, that include, fibroblasts, vascular endothelial cells, and/or activated T-cells and extra-cellular matrix components-with a modular perfusion system that uses a negative pressure chamber to draw fluid through the culture chamber and assay workspace without disturbing the positioning of the experiments. Real-time imaging in these 3D assays is performed in situ using confocal microscopy under controlled perfusion. In this study we present preliminary results from in situ studies of drug cytotoxicity, angiogenesis, and fibroblast activation, as well as, T cell migration and infiltration in bio-fabricated patient derived 3D tumor microtissues of OS and GB. We assessed the impact of oxygen tension, DNA damage, ER stress, and stromal composition (matrix and cell types) on response to frontline therapies, tumor angiogenesis, and T cell activation. Secretion of cytokines, pro-inflammatory factors and the release of damage-associated molecular patterns by microtumor constructs cultured under each condition was measured by ELISA. These data highlight the unique capabilities of our integrated confocal microscope and 3D passive perfusion culture system for studies of cancer biology, immunotherapy, and drug treatment regimens to provide in situ measurements of tumor-stroma interactions and immune cell invasion dynamics in precision fabricated OS and GB patient derived 3D microtissues. Citation Format: Padraic P. Levings, Juan M. Urueña, Eric O. McGhee, Alex J. McGhee, Derek L. Hood, Kylie E. Van Meter, Steve H. Ghivizzani, Colin J. Anderson, Wallace G. Sawyer. Patient-derived 3-dimensional models of pediatric tumors for in situ study of cancer biology, immunotherapy, and drug treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2887.

Identification of a nanostring signature that differentiates early pancreatic cancers according to stromal composition and predicts clinical outcome

Identification of a nanostring signature that differentiates early pancreatic cancers according to stromal composition and predicts clinical outcome

Abstract 2744: Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer

Abstract Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T-lymphocytes (CTLs). Unbiased analysis of the TCGA database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of CXCL12/CXCR4 axis, we inhibited CXCR4 and found that CXCR4 blockade decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. We confirmed that these immunosuppressive effects are dependent on cancer-associated fibroblast (CAF) signaling through genetic deletion of Cxcr4 in aSMA+ cells. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients. Citation Format: Ivy X. Chen, Vikash P. Chauhan, Jessica Posada, Mei R. Ng, Michelle Wu, Pichet Adstamongkonkul, Peigen Huang, Neal Lindeman, Robert Langer, Rakesh K. Jain. Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2744.

Extracellular vesicles impose quiescence on residual hematopoietic stem cells in the leukemic niche.

Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC.

A Novel TNM Assessing System for Predicting the Prognosis and Immunotherapy Response of Gastric Cancer

Background: The classical diagnostic method for the prognosis prediction of gastric cancer is imprecise. Thus, tools that can precisely predict the prognosis of gastric cancer subtypes and guide treatment strategies are needed. Methods: Using publicly available data from The Cancer Genome Atlas and Gene Expression Omnibus, we established tumor-noncoding RNA-microenvironment type (TNM) scores to define a combination of signatures that reflect the expression levels of tumor-related protein-coding genes (tumor score), tumor-related noncoding genes (NC score), and the cellular and stromal composition of the tumor microenvironment (TME score). These signatures were used to predict the prognosis of gastric cancer patients and the therapeutic effect of immunotherapy on gastric cancer. Results: Based on TNM scores, gastric cancer patients were divided into three subgroups and Kaplan-Meier survival curves revealed significant differences among the subgroups (P < 0.001). Tumor score was positively associated with the epithelial-mesenchymal transition signaling pathway and transforming growth factor-beta signaling pathway and negatively associated with DNA damage repair. The TME score showed opposite results and was also positively associated with cytotoxic T-cells and immune check-points. We also explored the predictive ability of TNM scores for the effectiveness of anti-PD-1/PD-L1 treatment. Receiver operating characteristic curves showed that a combination of TME score, tumor score, and tumor mutation burden could predict the treatment effect well (area under the curve = 0.708). Conclusion: Our study showed that the TNM scores could be used as a robust predicting tool for the prognosis of gastric cancer and the treatment effects of check-point inhibitors. Funding: This study was supported by the National Natural Science Foundation of China (No. 81772580, and 81811530024 to Wangjun Liao); Guangzhou Planed Project of Science and Technology (No. 201803010070 to Wangjun Liao). Declaration of Interest: The authors declare that they have no competing interests.

Integrative analysis identifies lincRNAs up- and downstream of neuroblastoma driver genes

Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN, ALK and PHOX2B. Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.